Conjugates and compositions for cellular delivery

ABSTRACT

This invention features conjugates, degradable linkers, compositions, methods of synthesis, and applications thereof, including cholesterol, folate, galactose, galactosamine, N-acetyl galactosamine, PEG, phospholipid, peptide and human serum albumin (HSA) derived conjugates of biologically active compounds, including antibodies, antivirals, chemotherapeutics, peptides, proteins, hormones, nucleosides, nucleotides, non-nucleosides, and nucleic acids including enzymatic nucleic acids, DNAzymes, allozymes, antisense, dsRNA, siNA, siRNA, triplex oligonucleotides, 2,5-A chimeras, decoys and aptamers.

[0001] This patent application is a continuation-in-part of Vargeese etal., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, which is acontinuation-in-part of Adamic et al., PCT/US02/15876, filed May 17,2002, that claims the benefit of Adamic et al., U.S. Ser. No.60/292,217, filed May 18, 2001, from Adamic et al., U.S. Ser. No.60/362,016 filed Mar. 6, 2002, both entitled ‘CONJUGATES ANDCOMPOSITIONS FOR CELLULAR DELIVERY’, from Vargeese et al., U.S. Ser. No.60/306,883, filed Jul. 20, 2001 entitled “CONJUGATES AND COMPOSITIONSFOR TRANSPORT ACROSS CELLULAR MEMBRANES”, and Vargeese et al., U.S. Ser.No. 60/311,865, filed Aug. 13, 2001, entitled “CONJUGATES ANDCOMPOSITIONS FOR CELLULAR DELIVERY”; and is also a continuation-in-partof McSwiggen et al., PCT/US03/05346, filed Feb. 20, 2003, and McSwiggenet al., PCT/US03/05028, filed Feb. 20, 2003, both of which claim thebenefit of Beigelman et al., U.S. Ser. No. 60/358,580 filed Feb. 20,2002, of Beigelman et al., U.S. Ser. No. 60/363,124 filed Mar. 11, 2002,of Beigelman et al., U.S. Ser. No. 60/386,782 filed Jun. 6, 2002, ofBeigelman et al., U.S. Ser. No. 60/406,784 filed Aug. 29, 2002, ofBeigelman et al., U.S. Ser. No. 60/408,378 filed Sep. 5, 2002, ofBeigelman et al., U.S. Ser. No. 60/409,293 filed Sep. 9, 2002, and ofBeigelman et al., U.S. Ser. No. 60/440,129 filed Jan. 15, 2003. Theseapplications are hereby incorporated by reference herein in theirentirety including the drawings.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to conjugates, compositions,methods of synthesis, and applications thereof. The discussion isprovided only for understanding of the invention that follows. Thissummary is not an admission that any of the work described below isprior art to the claimed invention.

[0003] The cellular delivery of various therapeutic compounds, such asantiviral and chemotherapeutic agents, is usually compromised by twolimitations. First the selectivity of therapeutic agents is often low,resulting in high toxicity to normal tissues. Secondly, the traffickingof many compounds into living cells is highly restricted by the complexmembrane systems of the cell. Specific transporters allow the selectiveentry of nutrients or regulatory molecules, while excluding mostexogenous molecules such as nucleic acids and proteins. Variousstrategies can be used to improve transport of compounds into cells,including the use of lipid carriers and various conjugate systems.Conjugates are often selected based on the ability of certain moleculesto be selectively transported into specific cells, for example viareceptor mediated endocytosis. By attaching a compound of interest tomolecules that are actively transported across the cellular membranes,the effective transfer of that compound into cells or specific cellularorganelles can be realized. Alternately, molecules that are able topenetrate cellular membranes without active transport mechanisms, forexample, various lipophilic molecules, can be used to deliver compoundsof interest. Examples of molecules that can be utilized as conjugatesinclude but are not limited to peptides, hormones, fatty acids,vitamins, flavonoids, sugars, reporter molecules, reporter enzymes,chelators, porphyrins, intercalcators, and other molecules that arecapable of penetrating cellular membranes, either by active transport orpassive transport.

[0004] The delivery of compounds to specific cell types, for example,cancer cells or cells specific to particular tissues and organs, can beaccomplished by utilizing receptors associated With specific cell types.Particular receptors are overexpressed in certain cancerous cells,including the high affinity folic acid receptor. For example, the highaffinity folate receptor is a tumor marker that is overexpressed in avariety of neoplastic tissues, including breast, ovarian, cervical,colorectal, renal, and nasoparyngeal tumors, but is expressed to a verylimited extent in normal tissues. The use of folic acid based conjugatesto transport exogenous compounds across cell membranes can provide atargeted delivery approach to the treatment and diagnosis of disease andcan provide a reduction in the required dose of therapeutic compounds.Furthermore, therapeutic bioavialability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates, including folate bioconjugates. Godwin et al., 1972, J.Biol. Chem., 247, 2266-2271, report the synthesis of biologically activepteroyloligo-L-glutamates. Habus et al., 1998, Bioconjugate Chem., 9,283-291, describe a method for the solid phase synthesis of certainoligonucleotide-folate conjugates. Cook, U.S. Pat. No. 6,721,208,describes certain oligonucleotides modified with specific conjugategroups. The use of biotin and folate conjugates to enhance transmembranetransport of exogenous molecules, including specific oligonucleotideshas been reported by Low et al., U.S. Pat. Nos. 5,416,016, 5,108,921,and International PCT publication No. WO 90/12096. Manoharan et al.,International PCT publication No. WO 99/66063 describe certain folateconjugates, including specific nucleic acid folate conjugates with aphosphoramidite moiety attached to the nucleic acid component of theconjugate, and methods for the synthesis of these folate conjugates.Nomura et al., 2000, J. Org. Chem., 65, 5016-5021, describe thesynthesis of an intermediate,alpha-[2-(trimethylsilyl)ethoxycarbonl]folic acid, useful in thesynthesis of ceratin types of folate-nucleoside conjugates. Guzaev etal., U.S. Pat. No. 6,335,434, describes the synthesis of certain folateoligonucleotide conjugates.

[0005] The delivery of compounds to other cell types can be accomplishedby utilizing receptors associated with a certain type of cell, such ashepatocytes. For example, drug delivery systems utilizingreceptor-mediated endocytosis have been employed to achieve drugtargeting as well as drug-uptake enhancement. The asialoglycoproteinreceptor (ASGPr) (see for example Wu and Wu, 1987, J. Biol. Chem. 262,4429-4432) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).Binding of such glycoproteins or synthetic glycoconjugates to thereceptor takes place with an affinity that strongly depends on thedegree of branching of the oligosaccharide chain, for example,triatennary structures are bound with greater affinity than biatenarryor monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620;Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987,Glycoconjugate J., 4, 317-328, obtained this high specificity throughthe use of N-acetyl-D-galactosamine as the carbohydrate moiety, whichhas higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease such as HBV and HCV infection or hepatocellularcarcinoma. The use of bioconjugates can also provide a reduction in therequired dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavialability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates.

[0006] A number of peptide based cellular transporters have beendeveloped by several research groups. These peptides are capable ofcrossing cellular membranes in vitro and in vivo with high efficiency.Examples of such fusogenic peptides include a 16-amino acid fragment ofthe homeodomain of ANTENNAPEDIA, a Drosophila transcription factor (Wanget al., 1995, PNAS USA., 92, 3318-3322); a 17-mer fragment representingthe hydrophobic region of the signal sequence of Kaposi fibroblastgrowth factor with or without NLS domain (Antopolsky et al., 1999,Bioconj. Chem., 10, 598-606); a 17-mer signal peptide sequence of caimancrocodylus Ig(5) light chain (Chaloin et al., 1997, Biochem. Biophys.Res. Comm., 243, 601-608); a 17-amino acid fusion sequence of HIVenvelope glycoprotein gp4114, (Morris et al., 1997, Nucleic Acids Res.,25, 2730-2736); the HIV-1 Tat49-57 fragment (Schwarze et al., 1999,Science, 285, 1569-1572); a transportan A—achimeric 27-mer consisting ofN-terminal fragment of neuropeptide galanine and membrane interactingwasp venom peptide mastoporan (Lindgren et al., 2000, BioconjugateChem., 11, 619-626); and a 24-mer derived from influenza virushemagglutinin envelop glycoprotein (Bongartz et al., 1994, Nucleic AcidsRes., 22, 4681-4688).

[0007] These peptides were successfully used as part of an antisenseoligonucleotide-peptide conjugate for cell culture transfection withoutlipids. In a number of cases, such conjugates demonstrated better cellculture efficacy then parent oligonucleotides transfected using lipiddelivery. In addition, use of phage display techniques has identifiedseveral organ targeting and tumor targeting peptides in vivo (Ruoslahti,1996, Ann. Rev. Cell Dev. Biol., 12, 697-715). Conjugation of tumortargeting peptides to doxorubicin has been shown to significantlyimprove the toxicity profile and has demonstrated enhanced efficacy ofdoxorubicin in the in vivo murine cancer model MDA-MB-435 breastcarcinoma (Arap et al., 1998, Science, 279, 377-380).

[0008] Hudson et al., 1999, Int. J. Pharm., 182, 49-58, describes thecellular delivery of specific hammerhead ribozymes conjugated to atransferrin receptor antibody. Janjic et al., U.S. Pat. No. 6,168,778,describes specific VEGF nucleic acid ligand complexes for targeted drugdelivery. Bonora et al., 1999, Nucleosides Nucleotides, 18, 1723-1725,describes the biological properties of specific antisenseoligonucleotides conjugated to certain polyethylene glycols. Davis andBishop, International PCT publication No. WO 99/17120 and Jaeschke etal., 1993, Tetrahedron Lett., 34, 301-4 describe specific methods ofpreparing polyethylene glycol conjugates. Tullis, International PCTPublication No. WO 88/09810; Jaschke, 1997, ACS Sympl Ser., 680,265-283; Jaschke et al., 1994, Nucleic Acids Res., 22, 4810-17; Efimovet al., 1993, Bioorg. Khim., 19, 800-4; and Bonora et al., 1997,Bioconjugate Chem., 8, 793-797, describe specific oligonucleotidepolyethylene glycol conjugates. Manoharan, International PCT PublicationNo. WO 00/76554, describes the preparation of specific ligand-conjugatedoligodeoxyribonucleotides with certain cellular, serum, or vascularproteins. Defrancq and Lhomme, 2001, Bioorg Med Chem Lett., 11, 931-933;Cebon et al., 2000, Aust. J. Chem., 53, 333-339; and Salo et al., 1999,Bioconjugate Chem., 10, 815-823 describe specific aminooxy peptideoligonucleotide conjugates.

SUMMARY OF THE INVENTION

[0009] The present invention features compositions and conjugates tofacilitate delivery of molecules into a biological system, such ascells. The conjugates provided by the instant invention can imparttherapeutic activity by transferring therapeutic compounds acrosscellular membranes. The present invention encompasses the design andsynthesis of novel agents for the delivery of molecules, including butnot limited to small molecules, lipids, nucleosides, nucleotides,nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins,negatively charged polymers and other polymers, for example proteins,peptides, hormones, carbohydrates, or polyamines, across cellularmembranes. In general, the transporters described are designed to beused either individually or as part of a multi-component system, with orwithout degradable linkers.

[0010] The compounds of the invention generally shown in the Formulaebelow are expected to improve delivery of molecules into a number ofcell types originating from different tissues, in the presence orabsence of serum.

[0011] The present invention features a compound having the Formula 1:

[0012] wherein each R₁, R₃, R₄, R₅, R₆, R₇ and R₈ is independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, or aprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and R₂ is a phosphorus containing group,nucleoside, nucleotide, small molecule, nucleic acid, polynucleotide, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, siNA or a portion thereof, or a solid supportcomprising a linker. The present invention features a compound havingthe Formula 2:

[0013] wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and R₂ is a phosphorus containing group, nucleoside,nucleotide, small molecule, nucleic acid, polynucleotide, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, siNA or a portion thereof, or a solid supportcomprising a linker.

[0014] The present invention features a compound having the Formula 3:

[0015] wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen,alkyl substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and R₂ is a phosphorus containing group, nucleoside,nucleotide, small molecule, or nucleic acid, polynucleotide, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, siNA or a portion thereof,.

[0016] The present invention features a compound having the Formula 4:

[0017] wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₂ is aphosphorus containing group, nucleoside, nucleotide, small molecule,nucleic acid, polynucleotide, or oligonucleotide such as an enzymaticnucleic acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,aptamer or triplex forming oligonucleotide, siNA or a portion thereof,or a solid support comprising a linker, and R₁₃ is an amino acid sidechain.

[0018] The present invention features a compound having the Formula 5:

[0019] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each R₃, R₅, R₆, R₇ and R₈ is independently hydrogen, alkyl ornitrogen protecting group, each “n” is independently an integer from 0to about 200, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each R₉ and R₁₀ is independently anitrogen containing group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

[0020] The present invention features a compound having the Formula 6:

[0021] wherein each R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, R₂ isa phosphorus containing group, nucleoside, nucleotide, small molecule,nucleic acid, polynucleotide, or oligonucleotide such as an enzymaticnucleic acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,aptamer or triplex forming oligonucleotide, siNA or a portion thereof,or a solid support comprising a linker, each “n” is independently aninteger from 0 to about 200, and L is a degradable linker.

[0022] The present invention features a compound having the Formula 7:

[0023] wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen,alkyl substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and R₂ is a phosphorus containing group, nucleoside,nucleotide, small molecule, nucleic acid, polynucleotide, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, siNA or a portion thereof, or a solid supportcomprising a linker.

[0024] The present invention features a compound having the Formula 8:

[0025] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl ornitrogen protecting group, each “n” is independently an integer from 0to about 200, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each R₉ and R₁₀ is independently anitrogen containing group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

[0026] The present invention features a method for synthesizing acompound having Formula 5:

[0027] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl ornitrogen protecting group, each “n” is independently an integer from 0to about 200, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each R₉ and R₁₀ is independently anitrogen containing group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group,comprising: coupling a bis-hydroxy aminoalkyl derivative, for exampleD-threoninol, with a N-protected aminoalkanoic acid to yield a compoundof Formula 9;

[0028] wherein R₁₁ is an amino protecting group, R₁₂ is a straight orbranched chain alkyl, substituted alkyl, aryl, or substituted aryl, andeach “n” is independently an integer from 0 to about 200; introducingprimary hydroxy protection R₁ followed by amino deprotection of R₁₁ toyield a compound of Formula 10;

[0029] wherein R₁ is a protecting group, R₁₂ is a straight or branchedchain alkyl, substituted alkyl, aryl, or substituted aryl, and each “n”is independently an integer from 0 to about 200; coupling thedeprotected amine of Formula 10 with a protected amino acid, for exampleglutamic acid, to yield a compound of Formula 11;

[0030] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each “n” is independently an integer from 0 to about 200, R₁₁is an amino protecting group, and R₁₂ is a straight or branched chainalkyl, substituted alkyl, aryl, or substituted aryl; deprotecting theamine R₁₁ of the conjugated glutamic acid of Formula XI to yield acompound of Formula 12;

[0031] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each “n” is independently an integer from 0 to about 200, R₁₁is an amino protecting group, and R₁₂ is a straight or branched chainalkyl, substituted alkyl, aryl, or substituted aryl; coupling thedeprotected amine of Formula 12 with an amino protected pteroic acid toyield a compound of Formula 13;

[0032] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl ornitrogen protecting group, R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl, and each “n” isindependently an integer from 0 to about 200; and introducing aphosphorus containing group at the secondary hydroxyl of Formula 13 toyield a compound of Formula 5.

[0033] The present invention features a method for synthesizing acompound having Formula 8:

[0034] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl ornitrogen protecting group, each “n” is independently an integer from 0to about 200, each R₉ and R₁₀ is independently a nitrogen containinggroup, cyanoalkoxy, alkoxy, aryloxy, or alkyl group, and R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, comprising; coupling a bis-hydroxy aminoalkylderivative, for example D-threoninol, with a protected amino acid, forexample glutamic acid, to yield a compound of Formula 14;

[0035] wherein R₁₁ is an amino protecting group, each “n” isindependently an integer from 0 to about 200, R₄ is independently aprotecting group, and R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl; introducing primaryhydroxy protection R₁ followed by amino deprotection of R₁₁ of Formula14 to yield a compound of Formula 15;

[0036] wherein each R₁ and R₄ is independently a protecting group orhydrogen, R₁₂ is a straight or branched chain alkyl, substituted alkyl,aryl, or substituted aryl, and each “n” is independently an integer from0 to about 200; coupling the deprotected amine of Formula 15 with anamino protected pteroic acid to yield a compound of Formula 16;

[0037] wherein each R₁ and R₄ is independently a protecting group orhydrogen, each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl ornitrogen protecting group, R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl, and each “n” isindependently an integer from 0 to about 200; and introducing aphosphorus containing group at the secondary hydroxyl of Formula 16 toyield a compound of Formula 8.

[0038] In one embodiment, R₂ of a compound of the invention comprises aphosphorus containing group.

[0039] In another embodiment, R₂ of a compound of the inventioncomprises a nucleoside, for example, a nucleoside with beneficialactivity such as anticancer or antiviral activity.

[0040] In yet another embodiment, R₂ of a compound of the inventioncomprises a nucleotide, for example, a nucleotide with beneficialactivity such as anticancer or antiviral activity.

[0041] In a further embodiment, R₂ of a compound of the inventioncomprises a small molecule, for example, a small molecule withbeneficial activity such as anticancer or antiviral activity.

[0042] In another embodiment, R₂ of a compound of the inventioncomprises a nucleic acid, polynucleotide, or oligonucleotide, forexample, a nucleic acid, polynucleotide, or oligonucleotide withbeneficial activity such as anticancer or antiviral activity.Non-limiting examples of nucleic acid, polynucleotide, andoligonucleotide compounds include enzymatic nucleic acid molecules,antisense molecules, aptamers, triplex forming oligonucleotides, decoys,2,5-A chimera molecules, and siNA or a portion thereof.

[0043] In one embodiment, R₂ of a compound of the invention comprises asolid support comprising a linker.

[0044] In another embodiment, a nucleoside (R₂) of the inventioncomprises a nucleoside with anticancer activity.

[0045] In another embodiment, a nucleoside (R₂) of the inventioncomprises a nucleoside with antiviral activity.

[0046] In another embodiment, the nucleoside (R₂) of the inventioncomprises fludarabine, lamivudine (3TC), 5-fluro uridine, AZT,ara-adenosine, ara-adenosine monophosphate, a dideoxy nucleoside analog,carbodeoxyguanosine, ribavirin, fialuridine, lobucavir, a pyrophosphatenucleoside analog, an acyclic nucleoside analog, acyclovir,gangciclovir, penciclovir, famciclovir, an L-nucleoside analog, FTC,L-FMAU, L-ddC, L-FddC, L-d4C, L-Fd4C, an L-dideoxypurine nucleosideanalog, cytallene, bis-POM PMEA (GS-840), BMS-200,475, carbovir orabacavir.

[0047] In one embodiment, R₁₃ of a compound of the invention comprisesan alkylamino or an alkoxy group, for example, —CH₂O— or —CH(CH₂)CH₂O—.

[0048] In another embodiment, R₁₂ of a compound of the invention is analkylhyrdroxyl, for example, —(CH₂)_(n)OH, where n comprises an integerfrom about 1 to about 10.

[0049] In another embodiment, L of Formula 6 of the invention comprisesserine, threonine, or a photolabile linkage.

[0050] In one embodiment, R₉ of a compound of the invention comprises aphosphorus protecting group, for example —OCH₂CH₂CN (oxyethylcyano).

[0051] In one embodiment, R₁₀ of a compound of the invention comprises anitrogen containing group, for example, —N(R₁₄) wherein R₁₄ is astraight or branched chain alkyl having from about 1 to about 10carbons.

[0052] In another embodiment, R₁₀ of a compound of the inventioncomprises a heterocycloalkyl or heterocycloalkenyl ring containing fromabout 4 to about 7 atoms, and having from about 1 to about 3 heteroatomscomprising oxygen, nitrogen, or sulfur.

[0053] In another embodiment, R₁ of a compound of the inventioncomprises an acid labile protecting group, such as a trityl orsubstituted trityl group, for example, a dimethoxytrityl ormono-methoxytrityl group.

[0054] In another embodiment, R₄ of a compound of the inventioncomprises a tert-butyl, Fm (fluorenyl-methoxy), or allyl group.

[0055] In one embodiment, R₆ of a compound of the invention comprises aTFA (trifluoracetyl) group.

[0056] In another embodiment, R₃, R₅, R₇ and R₈ of a compound of theinvention are independently hydrogen.

[0057] In one embodiment, R₇ of a compound of the invention isindependently isobutyryl, dimethylformamide, or hydrogen.

[0058] In another embodiment, R₁₂ of a compound of the inventioncomprises a methyl group or ethyl group.

[0059] In one embodiment, a nucleic acid of the invention comprises asiNA molecule or a portion thereof.

[0060] In one embodiment, a nucleic acid of the invention comprises anenzymatic nucleic acid, for example a hammerhead, Inozyme, DNAzyme,G-cleaver, Zinzyme, Amberzyme, or allozyme or a portion thereof.

[0061] In another embodiment, a nucleic acid of the invention comprisesan antisense nucleic acid, 2-5A nucleic acid chimera, or decoy nucleicacid or a portion thereof.

[0062] In another embodiment, the solid support having a linker of theinvention comprises a structure of Formula 17:

[0063] wherein SS is a solid support, and each “n” is independently aninteger from about 1 to about 200.

[0064] In another embodiment, the solid support of the instant inventionis controlled pore glass (CPG) or polystyrene, and can be used in thesynthesis of a nucleic acid, polynucleotide, or oligonucleotide or theinvention, such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer, triplex formingoligonucleotide, siNA or a portion thereof.

[0065] In one embodiment, the invention features a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable carrier.

[0066] In another embodiment, the invention features a method oftreating a cancer patient, comprising contacting cells of the patientwith a pharmaceutical composition of the invention under conditionssuitable for the treatment. This treatment can comprise the use of oneor more other drug therapies under conditions suitable for thetreatment. The cancers contemplated by the instant invention include butare not limited to breast cancer, lung cancer, colorectal cancer, braincancer, esophageal cancer, stomach cancer, bladder cancer, pancreaticcancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma,lymphoma, glioma, or multidrug resistant cancers.

[0067] In one embodiment, the invention features a method of treating apatient infected with a virus, comprising contacting cells of thepatient with a pharmaceutical composition of the invention, underconditions suitable for the treatment. This treatment can comprise theuse of one or more other drug therapies under conditions suitable forthe treatment. The viruses contemplated by the instant invention includebut are not limited to HIV, HBV, HCV, CMV, RSV, HSV, poliovirus,influenza, rhinovirus, west nile virus, Ebola virus, foot and mouthvirus, and papilloma virus.

[0068] In one embodiment, the invention features a kit for detecting thepresence of a nucleic acid molecule or other target molecule in asample, for example, a gene in a cancer cell, comprising a compound ofthe instant invention.

[0069] In one embodiment, the invention features a kit for detecting thepresence of a nucleic acid molecule, or other target molecule in asample, for example, a gene in a virus-infected cell, comprising acompound of the instant invention.

[0070] In another embodiment, the invention features a compound of theinstant invention comprising a modified phosphate group, for example, aphosphoramidite, phosphodiester, phosphoramidate, phosphorothioate,phosphorodithioate, alkylphosphonate, arylphosphonate, monophosphate,diphosphate, triphosphate, or pyrophosphate.

[0071] In one embodiment, the invention features a method forsynthesizing a compound having Formula 18:

[0072] wherein each R₆ and R₇ is independently hydrogen, alkyl ornitrogen protecting group, comprising: reacting folic acid with acarboxypeptidase to yield a compound of Formula 19;

[0073] introducing a protecting group R₆ on the secondary amine ofFormula 19 to yield a compound of Formula 20;

[0074] wherein R₆ is a nitrogen protecting group; and introducing aprotecting group R₇ on the primary amine of Formula 20 to yield acompound of Formula 18.

[0075] In another embodiment, the amino protected pteroic acid of theinvention is a compound of Formula 18.

[0076] In one embodiment, the invention encompasses a compound ofFormula 1 having Formula 21:

[0077] wherein each “n” is independently an integer from 0 to about 200.

[0078] In another embodiment, the invention encompasses a compound ofFormula 7 having Formula 22:

[0079] wherein each “n” is independently an integer from 0 to about 200.

[0080] In another embodiment, the invention encompasses a compound ofFormula 4 having Formula 23:

[0081] wherein “n” is an integer from 0 to about 200.

[0082] In another embodiment, the invention encompasses a compound ofFormula 4 having Formula 24:

[0083] wherein “n” is an integer from 0 to about 200.

[0084] In another embodiment, the invention features a compound havingFormula 25:

[0085] wherein each R₅ and R₇ is independently hydrogen, alkyl or anitrogen protecting group, each R₁₅, R₁₆, R₁₇, and R₁₈ is independentlyO, S, alkyl, substituted alkyl, aryl, substituted aryl, or halogen, X₁is —CH(X_(1′)) or a group of Formula 38:

[0086] wherein R₄ is a protecting group and “n” is an integer from 0 toabout 200;

[0087] X_(1′) is the protected or unprotected side chain of a naturallyoccurring or non-naturally-occurring amino acid, X₂ is amide, alkyl, orcarbonyl containing linker or a bond, and X₃ is a degradable linkerwhich is optionally absent.

[0088] In another embodiment, the X₃ group of Formula 25 comprises agroup of Formula 26:

[0089] wherein R₄ is hydrogen or a protecting group, “n” is an integerfrom 0 to about 200 and R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl.

[0090] In yet another embodiment, R₄ of Formula 26 is hydrogen and R₁₂is methyl or hyrdogen.

[0091] In still another embodiment, the invention features a compoundhaving Formula 27:

[0092] wherein “n” is an integer from about 20 to about 20, R₄ is H or acationic salt, and R₂₄ is a sulfur containing leaving group, for examplea group comprising:

[0093] In another embodiment, the invention features a method forsynthesizing a compound having Formula 27 comprising:

[0094] (a) selective tritylation of the thiol of cysteamine underconditions suitable to yield a compound having Formula 28:

[0095] wherein “n” is an integer from about 0 to about 20 and R₁₉ is athiol protecting group;

[0096] (b) peptide coupling of the product of (a) with a compound havingFormula 29:

[0097] wherein R₂₀ is a carboxylic acid protecting group and R₂₁ is anamino protecting group, under conditions suitable to yield a compoundhaving Formula 30:

[0098] wherein “n” is an integer from about 0 to about 20, R₁₉ is athiol protecting group, R₂₀ is a carboxylic acid protecting group andR₂₁ is an amino protecting group;

[0099] (c) removing the amino protecting group R₂₁ of the product of (b)under conditions suitable to yield a compound having Formula 31:

[0100] wherein “n” is an integer from about 0 to about 20 and R₁₉ andR₂₀ are as described in (b);

[0101] (d) condensation of the product of (c) with a compound havingFormula 32:

[0102] wherein R₂₂ is an amino protecting group, under conditionssuitable to yield a compound having Formula 33:

[0103] wherein “n” is an integer from about 0 to about 20 and R₁₉ andR₂₀ are as described in (b) and R₂₂ is as described in (d);

[0104] (e) selective cleavage of R₂₂ from the product of (d) underconditions suitable to yield a compound having Formula 34:

[0105] wherein “n” is an integer from about 0 to about 20 and R₁₉ andR₂₀ are as described in (b);

[0106] (f) coupling the product of (e) with a compound having Formula35:

[0107] wherein R₂₃ is an amino protecting group under conditionssuitable to yield a compound having Formula 36:

[0108] wherein R₂₃ is an amino protecting group, “n” is an integer fromabout 0 to about 20 and R₁₉ and R₂₀ are as described in (b);

[0109] (g) deprotecting the product of (f) under conditions suitable toyield a compound having Formula 37.

[0110] wherein “n” is an integer from about 0 to about 20; and

[0111] (h) introducing a disulphide-based leaving group to the productof (g) under conditions suitable to yield a compound having Formula 27.

[0112] In one embodiment, the invention features a compound havingFormula 39:

[0113] wherein “n” is an integer from about 0 to about 20, X is anucleic acid, polynucleotide, or oligonucleotide such as an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, and P is a phosphoruscontaining group. In another embodiment, X comprises a siNA molecule ora portion thereof.

[0114] In another embodiment, the invention features a method forsynthesizing a compound having Formula 39, comprising:

[0115] (a) Coupling a thiol containing linker to a nucleic acid,polynucleotide or oligonucleotide under conditions suitable to yield acompound having Formula 40:

[0116] wherein “n” is an integer from about 0 to about 20, X is anucleic acid, polynucleotide, or oligonucleotide, and P is a phosphoruscontaining group; and

[0117] (b) coupling the product of (a) with a compound having Formula 37under conditions suitable to yield a compound having Formula 39.

[0118] In another embodiment, the thiol containing linker of theinvention is a compound having Formula 41:

[0119] wherein “n” is an integer from about 0 to about 20, P is aphosphorus containing group, for example a phosphine, phosphite, orphosphate, and R₂₄ is any alkyl, substituted alkyl, alkoxy, aryl,substituted aryl, alkenyl, substituted alkenyl, alkynyl, or substitutedalkynyl group with or without additional protecting groups.

[0120] In another embodiment, the conditions suitable to yield acompound having Formula 40 comprises reduction, for example usingdithiothreitol (DTT) or any equivalent disulphide reducing agent, of thedisulfide bond of a compound having Formula 42:

[0121] wherein “n” is an integer from about 0 to about 20, X is anucleic acid, polynucleotide, or oligonucleotide, P is a phosphoruscontaining group, and R₂₄ is any alkyl, substituted alkyl, alkoxy, aryl,substituted aryl, alkenyl, substituted alkenyl, alkynyl, or substitutedalkynyl group with or without additional protecting groups. In anotherembodiment, X comprises a siNA molecule or a portion thereof.

[0122] In one embodiment, the invention features a compound havingFormula 43:

[0123] wherein X comprises a biologically active molecule; W comprises adegradable nucleic acid linker; Y comprises a linker molecule or aminoacid that can be present or absent; Z comprises H, OH, O-alkyl, SH,S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino,substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, orlabel; n is an integer from about 1 to about 100; and N′ is an integerfrom about 1 to about 20. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, W is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0124] In another embodiment, the invention features a compound havingFormula 44:

[0125] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent; n isan integer from about 1 to about 50, and PEG represents a compoundhaving Formula 45:

[0126] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; and n is an integerfrom about 1 to about 100. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, W is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0127] In another embodiment, the invention features a compound havingFormula 46:

[0128] wherein X comprises a biologically active molecule; each Windependently comprises linker molecule or chemical linkage that can bepresent or absent, Y comprises a linker molecule or chemical linkagethat can be present or absent; and PEG represents a compound havingFormula 45:

[0129] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; and n is an integerfrom about 1 to about 100. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, W is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0130] In one embodiment, the invention features a compound havingFormula 47:

[0131] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe the same or different and can be present or absent, Y comprises alinker molecule that can be present or absent; each Q independentlycomprises a hydrophobic group or phospholipid; each R1, R2, R3, and R4independently comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and n is aninteger from about 1 to about 10. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0132] In another embodiment, the invention features a compound havingFormula 48:

[0133] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises 0, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and B represents a lipophilic group, for example asaturated or unsaturated linear, branched, or cyclic alkyl group,cholesterol, or a derivative thereof. In another embodiment, X comprisesa siNA molecule or a portion thereof. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0134] In another embodiment, the invention features a compound havingFormula 49:

[0135] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and Brepresents a lipophilic group, for example a saturated or unsaturatedlinear, branched, or cyclic alkyl group, cholesterol, or a derivativethereof. In another embodiment, X comprises a siNA molecule or a portionthereof. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

[0136] In another embodiment, the invention features a compound havingFormula 50:

[0137] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule or chemical linkage that can be present orabsent; and each Q independently comprises a hydrophobic group orphospholipid. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0138] In one embodiment, the invention features a compound havingFormula 51:

[0139] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent; Ycomprises a linker molecule or amino acid that can be present or absent;Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,substituted aryl, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, or label; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and n is aninteger from about 1 to about 20. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0140] In another embodiment, the invention features a compound havingFormula 52:

[0141] wherein X comprises a biologically active molecule; Y comprises alinker molecule or chemical linkage that can be present or absent; eachR1, R2, R3, R4, and R5 independently comprises 0, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N; Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; SG comprises a sugar,for example galactose, galactosamine, N-acetyl-galactosamine, glucose,mannose, fructose, or fucose and the respective D or L, alpha or betaisomers, n is an integer from about 1 to about 20; and N′ is an integerfrom about 1 to about 20. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, Y is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0142] In another embodiment, the invention features a compound havingFormula 53:

[0143] wherein B comprises H, a nucleoside base, or a non-nucleosidicbase with or without protecting groups; each R1 independently comprisesO, N, S, alkyl, or substituted N; each R2 independently comprises O, OH,H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; each R3 independently comprises N or O—N,each R4 independently comprises O, CH2, S, sulfone, or sulfoxy; Xcomprises H, a removable protecting group, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymatic nucleicacid, siNA or a portion thereof, amino acid, peptide, protein, lipid,phospholipid, or label; W comprises a linker molecule or chemicallinkage that can be present or absent; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,each n is independently an integer from about 1 to about 50; and N′ isan integer from about 1 to about 10. In another embodiment, X comprisesa siNA molecule or a portion thereof. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0144] In another embodiment, the invention features a compound havingFormula 54:

[0145] wherein B comprises H, a nucleoside base, or a non-nucleosidicbase with or without protecting groups; each R1 independently comprises0, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N,or a phosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, siNA or a portion thereof,amino acid, peptide, protein, lipid, phospholipid, or label; W comprisesa linker molecule or chemical linkage that can be present or absent; andSG comprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers. In another embodiment, Xcomprises a siNA molecule or a portion thereof. In another embodiment, Wis selected from the group consisting of amide, phosphate, phosphateester, phosphoramidate, or thiophosphate ester linkage.

[0146] In one embodiment, the invention features a compound havingFormula 55:

[0147] wherein each R1 independently comprises O, N, S, alkyl, orsubstituted N; each R2 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a phosphoruscontaining group; each R3 independently comprises H, OH, alkyl,substituted alkyl, or halo; X comprises H, a removable protecting group,amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, siNA or a portion thereof, 2,5-A chimera, decoy, aptameror triplex forming oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, biologically active molecule or label; W comprises alinker molecule or chemical linkage that can be present or absent; SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, each n is independently aninteger from about 1 to about 50; and N′ is an integer from about 1 toabout 100. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0148] In another embodiment, the invention features a compound havingFormula 56:

[0149] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide such as an enzymatic nucleicacid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, siNA or a portion thereof, amino acid,peptide, protein, lipid, phospholipid, biologically active molecule orlabel; W comprises a linker molecule or chemical linkage that can bepresent or absent; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and each n isindependently an integer from about 0 to about 20. In anotherembodiment, X comprises a siNA molecule or a portion thereof. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

[0150] In another embodiment, the invention features a compound havingFormula 57:

[0151] wherein R1 can include the groups:

[0152] and wherein R2 can include the groups:

[0153] and wherein Tr is a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl; SG comprises a sugar, forexample galactose, galactosamine, N-acetyl-galactosamine, glucose,mannose, fructose, or fucose and the respective D or L, alpha or betaisomers, and n is an integer from about 1 to about 20.

[0154] In one embodiment, compounds having Formula 52, 53, 54, 55, 56,and 57 are featured wherein each nitrogen adjacent to a carbonyl canindependently be substituted for a carbonyl adjacent to a nitrogen oreach carbonyl adjacent to a nitrogen can be substituted for a nitrogenadjacent to a carbonyl.

[0155] In another embodiment, the invention features a compound havingFormula 58:

[0156] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent; Ycomprises a linker molecule or amino acid that can be present or absent;V comprises a signal protein or peptide, for example Human serum albuminprotein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoproteingp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each n is independentlyan integer from about 1 to about 50; and N′ is an integer from about 1to about 100. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0157] In another embodiment, the invention features a compound havingFormula 59:

[0158] wherein each R1 independently comprises O, S, N, substituted N,or a phosphorus containing group; each R2 independently comprises O, S,or N; X comprises H, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, or enzymatic nucleic acid or otherbiologically active molecule; n is an integer from about 1 to about 50,Q comprises H or a removable protecting group which can be optionallyabsent, each W independently comprises a linker molecule or chemicallinkage that can be present or absent, and V comprises a signal proteinor peptide, for example Human serum albumin protein, Antennapediapeptide, Kaposi fibroblast growth factor peptide, Caiman crocodylusIg(5) light chain peptide, HIV envelope glycoprotein gp41 peptide, HIV-1Tat peptide, Influenza hemagglutinin envelope glycoprotein peptide, ortransportan A peptide, or a compound having Formula 45

[0159] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide such as anenzymatic nucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-Achimera, decoy, aptamer or triplex forming oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; and n is an integerfrom about 1 to about 100. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, W is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0160] In another embodiment, the invention features a compound havingFormula 60:

[0161] wherein R1 can include the groups:

[0162] and wherein R2 can include the groups:

[0163] and wherein Tr is a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl; n is an integer fromabout 1 to about 50; and R8 is a nitrogen protecting group, for examplea phthaloyl, trifluoroacetyl, FMOC, or monomethoxytrityl group.

[0164] In another embodiment, the invention features a compound havingFormula 61:

[0165] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe the same or different and can be present or absent, Y comprises alinker molecule that can be present or absent; each 5 independentlycomprises a signal protein or peptide, for example Human serum albuminprotein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoproteingp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and n is aninteger from about 1 to about 10. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0166] In another embodiment, the invention features a compound havingFormula 62:

[0167] wherein X comprises a biologically active molecule; each 5independently comprises a signal protein or peptide, for example Humanserum albumin protein, Antennapedia peptide, Kaposi fibroblast growthfactor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide; Wcomprises a linker molecule or chemical linkage that can be present orabsent; each R1, R2, and R3 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and each n is independently an integer from about 1 toabout 10. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0168] In another embodiment, the invention features a compound havingFormula 63:

[0169] wherein X comprises a biologically active molecule; V comprises asignal protein or peptide, for example Human serum albumin protein,Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caimancrocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; W comprises a linkermolecule or chemical linkage that can be present or absent; each R1, R2,R3 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, R4represents an ester, amide, or protecting group, and each n isindependently an integer from about 1 to about 10. In anotherembodiment, X comprises a siNA molecule or a portion thereof. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

[0170] In another embodiment, the invention features a compound havingFormula 64:

[0171] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises 0, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, A comprises a nitrogen containing group, and B comprisesa lipophilic group. In another embodiment, X comprises a siNA moleculeor a portion thereof. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

[0172] In another embodiment, the invention features a compound havingFormula 65:

[0173] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, RV comprises the lipid or phospholipid component of anyof Formulae 47-50, and R6 comprises a nitrogen containing group. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

[0174] In another embodiment, the invention features a compound havingFormula 92:

[0175] wherein B comprises H, a nucleoside base, or a non-nucleosidicbase with or without protecting groups; each R1 independently comprises0, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N,or a phosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, biologically active molecule or label; W comprisesa linker molecule or chemical linkage that can be present or absent; R2comprises O, NH, S, CO, COO, ON═C, or alkyl; R3 comprises alkyl, akloxy,or an aminoacyl side chain; and SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

[0176] In another embodiment, the invention features a compound havingFormula 86:

[0177] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid, aminoacid, peptide, protein, lipid, phospholipid, biologically activemolecule or label; W comprises a linker molecule or chemical linkagethat can be present or absent; R3 comprises O, NH, S, CO, COO, ON═C, oralkyl; R4 comprises alkyl, akloxy, or an aminoacyl side chain; and SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, and each n is independently aninteger from about 0 to about 20. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0178] In another embodiment, the invention features a compound havingFormula 87:

[0179] wherein X comprises a protein, peptide, antibody, lipid,phospholipid, oligosaccharide, label, biologically active molecule, forexample a vitamin such as folate, vitamin A, E, B6, B12, coenzyme,antibiotic, antiviral, nucleic acid, nucleotide, nucleoside, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, siNA, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, or polymers such as polyethylene glycol; W comprises alinker molecule or chemical linkage that can be present or absent; and Ycomprises a biologically active molecule, for example an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, peptide, protein, orantibody; R1 comprises H, alkyl, or substituted alkyl. In anotherembodiment, X comprises a siNA molecule or a portion thereof. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

[0180] In another embodiment, the invention features a compound havingFormula 88:

[0181] wherein X comprises a protein, peptide, antibody, lipid,phospholipid, oligosaccharide, label, biologically active molecule, forexample a vitamin such as folate, vitamin A, E, B6, B12, coenzyme,antibiotic, antiviral, nucleic acid, nucleotide, nucleoside, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, siNA, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, or polymers such as polyethylene glycol; W comprises alinker molecule or chemical linkage that can be present or absent, and Ycomprises a biologically active molecule, for example an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, peptide, protein, orantibody. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0182] In another embodiment, the invention features a compound havingFormula 99:

[0183] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises 0, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine or branched derivative thereof,glucose, mannose, fructose, or fucose and the respective D or L, alphaor beta isomers. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0184] In another embodiment, the invention features a compound havingFormula 100:

[0185] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine or branched derivative thereof, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

[0186] In one embodiment, the SG component of any compound havingFormulae 99 or 100 comprises a compound having Formula 101:

[0187] wherein Y comprises a linker molecule or chemical linkage thatcan be present or absent and each R7 independently is hydrogen or anacyl group, for example an acetyl group.

[0188] In one embodiment, the W-SG component of a compound havingFormulae 99 comprises a compound having Formula 102:

[0189] wherein R2 comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylhalo, S, N, substituted N, a protecting group, or anothercompound having Formula 102; R1 independently H, OH, alkyl, substitutedalkyl, or halo and each R7 independently is hydrogen or an acyl group,for example an acetyl group, and R3 comprises O or R3 in Formula 99, andn is an integer from about 1 to about 20.

[0190] In one embodiment, the W-SG component of a compound havingFormulae 99 comprises a compound having Formula 103:

[0191] wherein R1 comprises H, alkyl, alkylhalo, O-alkyl, O-alkylhalo,S, N, substituted N, a protecting group, or another compound havingFormula 103; each R7 independently is hydrogen or an acyl group, forexample an acetyl group, and R3 comprises H or R3 in Formula 99, andeach n is independently an integer from about 1 to about 20.

[0192] In one embodiment, the invention features a compound havingFormula 104:

[0193] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,and each n is independently an integer from about 1 to about 20, and

[0194] wherein R1 can include the groups:

[0195] and wherein R2 can include the groups:

[0196] In one embodiment, the invention features a compound havingFormula 105:

[0197] wherein X comprises a nucleotide, polynucleotide, oroligonucleotide or a portion thereof, R2 comprises 0, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, a protectinggroup, or a nucleotide, polynucleotide, or oligonucleotide or a portionthereof; R1 independently H, OH, alkyl, substituted alkyl, or halo andeach R7 independently is hydrogen or an acyl group, for example anacetyl group, and n is an integer from about 1 to about 20. In anotherembodiment, X comprises a siNA molecule or a portion thereof.

[0198] In one embodiment, the invention features a compound havingFormula 106:

[0199] wherein X comprises a nucleotide, polynucleotide, oroligonucleotide or a portion thereof, R1 comprises H, OH, amino,substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,label, or a portion thereof, or OR5 where R5 a removable protectinggroup, each R7 independently is hydrogen or an acyl group, for examplean acetyl group, and each n is independently an integer from about 1 toabout 20. In another embodiment, X comprises a siNA molecule or aportion thereof.

[0200] In another embodiment, the invention features a compound havingFormula 107:

[0201] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and Cholesterol comprises cholesterol or an analog,derivative, or metabolite thereof. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

[0202] In another embodiment, the invention features a compound havingFormula 108:

[0203] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, andCholesterol comprises cholesterol or an analog, derivative, ormetabolite thereof. In another embodiment, X comprises a siNA moleculeor a portion thereof. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

[0204] In one embodiment, the W-Cholesterol component of a compoundhaving Formula 107 comprises a compound having Formula 109:

[0205] wherein R3 comprises R3 as described in Formula 107, and n isindependently an integer from about 1 to about 20.

[0206] In one embodiment, the invention features a compound havingFormula 110:

[0207] wherein R4 comprises 0, alkyl, alkylhalo, O-alkyl, O-alkylcyano,S, S-alkyl, S-alkylcyano, N or substituted N, each n is independently aninteger from about 1 to about 20, and

[0208] wherein R1 can include the groups:

[0209] and wherein R2 can include the groups:

[0210] In one embodiment, the invention features a compound havingFormula 111:

[0211] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, and nis an integer from about 1 to about 20. In another embodiment, Xcomprises a siNA molecule or a portion thereof. In another embodiment, Wis selected from the group consisting of amide, phosphate, phosphateester, phosphoramidate, or thiophosphate ester linkage.

[0212] In one embodiment, the invention features a compound havingFormula 112:

[0213] wherein n is an integer from about 1 to about 20. In anotherembodiment, a compound having Formula 112 is used to generate a compoundhaving Formula 111 via NHS ester mediated coupling with a biologicallyactive molecule, such as a siNA molecule or a portion thereof. In anon-limiting example, the NHS ester coupling can be effectuated viaattachment to a free amine present in the siNA molecule, such as anamino linker molecule present on a nucleic acid sugar (e.g. 2′-aminolinker) or base (e.g., C5 alkyl amine linker) component of the siNAmolecule.

[0214] In one embodiment, the invention features a compound havingFormula 113:

[0215] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises 0, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n isindependently an integer from about 1 to about 20, and

[0216] wherein R1 can include the groups:

[0217] and wherein R2 can include the groups:

[0218] In another embodiment, a compound having Formula 113 is used togenerate a compound having Formula 111 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

[0219] In one embodiment, the invention features a compound havingFormula 114:

[0220] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, and nis an integer from about 1 to about 20. In another embodiment, Xcomprises a siNA molecule or a portion thereof. In another embodiment, Wis selected from the group consisting of amide, phosphate, phosphateester, phosphoramidate, or thiophosphate ester linkage.

[0221] In one embodiment, the invention features a compound havingFormula 115:

[0222] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, R3comprises H, OH, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, label, or a portion thereof, or OR5 where R5 a removableprotecting group, and each n is independently an integer from about 1 toabout 20. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

[0223] In one embodiment, the invention features a compound havingFormula 116:

[0224] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises 0, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n isindependently an integer from about 1 to about 20, and

[0225] wherein R1 can include the groups:

[0226] and wherein R2 can include the groups:

[0227] In another embodiment, a compound having Formula 116 is used togenerate a compound having Formula 114 or 115 via phosphoramiditemediated coupling with a biologically active molecule, such as a siNAmolecule or a portion thereof.

[0228] In one embodiment, the invention features a compound havingFormula 117:

[0229] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises 0, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,each n is independently an integer from about 1 to about 20, and

[0230] wherein R1 can include the groups:

[0231] and wherein R2 can include the groups:

[0232] In another embodiment, a compound having Formula 117 is used togenerate a compound having Formula 105 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

[0233] In one embodiment, the invention features a compound havingFormula 118:

[0234] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, R3comprises H, OH, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, label, or a portion thereof, or OR5 where R5 a removableprotecting group, each R7 independently is hydrogen or an acyl group,for example an acetyl group, and each n is independently an integer fromabout 1 to about 20. In another embodiment, X comprises a siNA moleculeor a portion thereof. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

[0235] In one embodiment, the invention features a compound havingFormula 119:

[0236] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, eachR7 independently is hydrogen or an acyl group, for example an acetylgroup, and each n is independently an integer from about 1 to about 20.In another embodiment, X comprises a siNA molecule or a portion thereof.In another embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

[0237] In one embodiment, the invention features a compound havingFormula 120:

[0238] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,each n is independently an integer from about 1 to about 20, and

[0239] wherein R1 can include the groups:

[0240] and wherein R2 can include the groups:

[0241] In another embodiment, a compound having Formula 120 is used togenerate a compound having Formula 118 or 119 via phosphoramiditemediated coupling with a biologically active molecule, such as a siNAmolecule or a portion thereof.

[0242] In one embodiment, the invention features a compound havingFormula 121:

[0243] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, eachR7 independently is hydrogen or an acyl group, for example an acetylgroup, and each n is independently an integer from about 1 to about 20.In another embodiment, X comprises a siNA molecule or a portion thereof.In another embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

[0244] In one embodiment, the invention features a compound havingFormula 122:

[0245] wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,each n is independently an integer from about 1 to about 20, and

[0246] wherein R1 can include the groups:

[0247] and wherein R2 can include the groups:

[0248] In another embodiment, a compound having Formula 122 is used togenerate a compound having Formula 121 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

[0249] In one embodiment, the invention features a method for thesynthesis of a compound having Formula 48:

[0250] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises 0, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N; and each B independently represents a lipophilic group,for example a saturated or unsaturated linear, branched, or cyclic alkylgroup, comprising: (a) introducing a compound having Formula 66:

[0251] wherein R1 is defined as in Formula 48 and can include thegroups:

[0252] and wherein R2 is defined as in Formula 48 and can include thegroups:

[0253] and wherein each R5 independently comprises O, N, or S and eachR6 independently comprises a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl group, to a compoundhaving Formula 67:

X—W—Y  67

[0254] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 68:

[0255] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; and each R1,R2, R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo,O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted Ncomprising, each R5 independently comprises O, S, or N; and each R6 isindependently a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl group; (b) removing R6 from thecompound having Formula 26 and (c) introducing a compound having Formula69:

[0256] wherein R1 is defined as in Formula 48 and can include thegroups:

[0257] and wherein R2 is defined as in Formula 48 and can include thegroups:

[0258] wherein W and B are defined as in Formula 48, to the compoundhaving Formula 68 under conditions suitable for the formation of acompound having Formula 48.

[0259] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 49:

[0260] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N; each R5independently comprises O, S, or N; and each B independently comprises alipophilic group, for example a saturated or unsaturated linear,branched, or cyclic alkyl group, comprising: (a) coupling a compoundhaving Formula 70:

[0261] wherein R1 is defined as in Formula 49 and can include thegroups:

[0262] and wherein R2 is defined as in Formula 49 and can include thegroups:

[0263] and wherein each R5 independently comprises O, S, or N, andwherein each B independently comprises a lipophilic group, for example asaturated or unsaturated linear, branched, or cyclic alkyl group, with acompound having Formula 67:

X—W—Y  67

[0264] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 49.

[0265] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 52:

[0266] wherein X comprises a biologically active molecule; Y comprises alinker molecule or chemical linkage that can be present or absent; eachR1, R2, R3, and R4 independently comprises 0, OH, H, alkyl, alkylhalo,O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N; Zcomprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,substituted aryl, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, or label; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, n is an integerfrom about 1 to about 20; and N′ is an integer from about 1 to about 20,comprising: (a) coupling a compound having Formula 71:

[0267] wherein R1, R2, R3, R5, SG, and n is as defined in Formula 52,and wherein R1 can include the groups:

[0268] and wherein R2 can include the groups:

[0269] and R6 comprises a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl group; with a compoundhaving Formula 72:

X—Y  72

[0270] wherein X comprises a biologically active molecule and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 95:

[0271] (b) removing R6 from the compound having Formula 95 and (c)optionally coupling a nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, orlabel, or optionally; coupling a compound having Formula 71 under andoptionally repeating (b) and (c) under conditions suitable for theformation of a compound having Formula 52.

[0272] In another embodiment, the invention features a method forsynthesizing a compound having Formula 53:

[0273] wherein B comprises H, a nucleoside base, or a non-nucleosidicbase with or without protecting groups; each R1 independently comprisesO, N, S, alkyl, or substituted N; each R2 independently comprises O, OH,H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; each R3 independently comprises N or O—N,each R4 independently comprises O, CH2, S, sulfone, or sulfoxy; Xcomprises H, a removable protecting group, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymatic nucleicacid, amino acid, peptide, protein, lipid, phospholipid, or label; Wcomprises a linker molecule or chemical linkage that can be present orabsent; SG comprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, each n is independently aninteger from about 1 to about 50; and N′ is an integer from about 1 toabout 10, comprising: coupling a compound having Formula 73:

[0274] wherein R1, R2, R3, R4, X, W, B, N′ and n are as defined inFormula 53, with a sugar, for example a compound having Formula 74:

[0275] wherein Y comprises a linker molecule or chemical linkage thatcan be present or absent; L represents a reactive chemical group, forexample a NHS ester, and each R7 independently is hydrogen or an acylgroup, for example an acetyl group; under conditions suitable for theformation of a compound having Formula 53.

[0276] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 54:

[0277] wherein B comprises H, a nucleoside base, or a non-nucleosidicbase with or without protecting groups; each R1 independently comprises0, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N,or a phosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, biologically active molecule or label; W comprisesa linker molecule or chemical linkage that can be present or absent; SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, comprising (a) coupling acompound having Formula 75:

[0278] wherein R1, R2, R3, R4, X, W, and B are as defined in Formula 53,with a sugar, for example a compound having Formula 74.

[0279] wherein Y comprises a C11 alkyl linker molecule; L represents areactive chemical group, for example a NHS ester, and each R7independently is hydrogen or an acyl group, for example an acetyl group;under conditions suitable for the formation of a compound having Formula54.

[0280] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 55:

[0281] wherein each R1 independently comprises O, N, S, alkyl, orsubstituted N; each R2 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or a phosphoruscontaining group; each R3 independently comprises H, OH, alkyl,substituted alkyl, or halo; X comprises H, a removable protecting group,nucleotide, nucleoside, nucleic acid, oligonucleotide, or enzymaticnucleic acid or biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent; SG comprisesa sugar, for example galactose, galactosamine, N-acetyl-galactosamine,glucose, mannose, fructose, or fucose and the respective D or L, alphaor beta isomers, each n is independently an integer from about 1 toabout 50; and N′ is an integer from about 1 to about 100, comprising:(a) coupling a compound having Formula 76:

[0282] wherein R1 can include the groups:

[0283] and wherein R2 can include the groups:

[0284] and wherein each R3 independently comprises H, OH, alkyl,substituted alkyl, or halo; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and n is aninteger from about 1 to about 20, to a compound X—W, wherein X comprisesa nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymaticnucleic acid, amino acid, peptide, protein, lipid, phospholipid,biologically active molecule or label, and W comprises a linker moleculeor chemical linkage that can be present or absent; and (b) optionallyrepeating step (a) under conditions suitable for the formation of acompound having Formula 55.

[0285] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 56:

[0286] wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid, aminoacid, peptide, protein, lipid, phospholipid, biologically activemolecule or label; W comprises a linker molecule or chemical linkagethat can be present or absent; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,and each n is independently an integer from about 0 to about 20,comprising: (a) coupling a compound having Formula 77:

[0287] wherein each R1, X, W, and n are as defined in Formula 56, to asugar, for example a compound having Formula 74:

[0288] wherein Y comprises an alkyl linker molecule of length n, where nis an integer from about 1 to about 20; L represents a reactive chemicalgroup, for example a NHS ester, and each R7 independently is hydrogen oran acyl group, for example an acetyl group; and (b) optionally couplingX—W, wherein X comprises a removable protecting group, amino,substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, or label and W comprises a linker molecule orchemical linkage that can be present or absent, under conditionssuitable for the formation of a compound having Formula 56.

[0289] In another embodiment, the invention features method forsynthesizing a compound having Formula 57:

[0290] wherein R1 can include the groups:

[0291] and wherein R2 can include the groups:

[0292] and wherein Tr is a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl; SG comprises a sugar, forexample galactose, galactosamine, N-acetyl-galactosamine, glucose,mannose, fructose, or fucose and the respective D or L, alpha or betaisomers, and n is an integer from about 1 to about 20, comprising: (a)coupling a compound having Formula 77:

[0293] wherein R1 and X comprise H, to a sugar, for example a compoundhaving Formula 74:

[0294] wherein Y comprises an alkyl linker molecule of length n, where nis an integer from about 1 to about 20; L represents a reactive chemicalgroup, for example a NHS ester, and each R7 independently is hydrogen oran acyl group, for example an acetyl group; and (b) introducing a tritylgroup, for example a dimethoxytrityl, monomethoxytrityl, or trityl groupto the primary hydroxyl of the product of (a) and (c) introducing aphosphorus containing group having Formula 78:

[0295] wherein R1 can include the groups:

[0296] and wherein each R2 and R3 independently can include the groups:

[0297] to the secondary hydroxyl of the product of (b) under conditionssuitable for the formation of a compound having Formula 57.

[0298] In another embodiment, the invention features a method forsynthesizing a compound having Formula 60:

[0299] wherein R1 can include the groups:

[0300] and wherein R2 can include the groups:

[0301] and wherein Tr is a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl; n is an integer fromabout 1 to about 50; and R8 is a nitrogen protecting group, for examplea phthaloyl, trifluoroacetyl, FMOC, or monomethoxytrityl group,comprising: (a) introducing carboxy protection to a compound havingFormula 79:

[0302] wherein n is an integer from about 1 to about 50, underconditions suitable for the formation of a compound having Formula 80:

[0303] wherein n is an integer from about 1 to about 50 and R7 is acarboxylic acid protecting group, for example a benzyl group; (b)introducing a nitrogen containing group to the product of (a) underconditions suitable for the formation of a compound having Formula 81:

[0304] wherein n and R7 are as defined in Formula 80 and R8 is anitrogen protecting group, for example a phthaloyl, trifluoroacetyl,FMOC, or monomethoxytrityl group; (c) removing the carboxylic acidprotecting group from the product of (b) and introducingaminopropanediol under conditions suitable for the formation of acompound having Formula 82:

[0305] wherein n and R8 are as defined in Formula 81; (d) introducing aremovable protecting group, for example a trityl, monomethoxytrityl, ordimethoxytrityl to the product of (c) under conditions suitable for theformation of a compound having Formula 83:

[0306] wherein Tr, n and R8 are as defined in Formula 60; and (e)introducing a phosphorus containing group having Formula 78:

[0307] wherein R1 can include the groups:

[0308] and wherein each R2 and R3 independently can include the groups:

[0309] to the product of (d) under conditions suitable for the formationof a compound having Formula 60.

[0310] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 59:

[0311] wherein each R1 independently comprises O, S, N, substituted N,or a phosphorus containing group; each R2 independently comprises O, S,or N; X comprises H, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, such as an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide or other biologically active molecule ora portion thereof; n is an integer from about 1 to about 50, Q comprisesH or a removable protecting group which can be optionally absent, each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, and V comprises a protein or peptide, for exampleHuman serum albumin protein, Antennapedia peptide, Kaposi fibroblastgrowth factor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide,or a compound having Formula 45:

[0312] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; and n is an integerfrom about 1 to about 100, comprising: (a) removing R8 from a compoundhaving Formula 84:

[0313] wherein Q, X, W, R1, R2, and n are as defined in Formula 59 andR8 is a nitrogen protecting group, for example a phthaloyl,trifluoroacetyl, FMOC, or monomethoxytrityl group, under conditionssuitable for the formation of a compound having Formula 85:

[0314] wherein Q, X, W, R1, R2, and n are as defined in Formula 59; (b)introducing a group V to the product of (a) via the formation of anoxime linkage, wherein V comprises a protein or peptide, for exampleHuman serum albumin protein, Antennapedia peptide, Kaposi fibroblastgrowth factor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide,or a compound having Formula 45:

[0315] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; and n is an integerfrom about 1 to about 100, under conditions suitable for the formationof a compound having Formula 59.

[0316] In another embodiment, the invention features a method forsynthesizing a compound having Formula 64:

[0317] wherein X comprises a biologically active molecule; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, A comprises a nitrogen containing group, and B comprisesa lipophilic group, comprising: (a) introducing a compound havingFormula 66:

[0318] wherein R1 is defined as in Formula 64 and can include thegroups:

[0319] and wherein R2 is defined as in Formula 64 and can include thegroups:

[0320] and wherein each R5 independently comprises O, N, or S and eachR6 independently comprises a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl group, to a compoundhaving Formula 67:

X—W—Y  67

[0321] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 68:

[0322] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; and each R1,R2, R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo,O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted Ncomprising, each R5 independently comprises O, S, or N; and each R6 isindependently a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl group; (b) removing R6 from thecompound having Formula 68 and (c) introducing a compound having Formula69:

[0323] wherein R1 is defined as in Formula 64 and can include thegroups:

[0324] and wherein R2 is defined as in Formula 64 and can include thegroups:

[0325] and wherein R3, W and B are defined as in Formula 64; andintroducing a compound having Formula 69′:

[0326] wherein R1 is defined as in Formula 64 and can include thegroups:

[0327] and wherein R2 is defined as in Formula 48 and can include thegroups:

[0328] and wherein R3, W and A are defined as in Formula 64; to thecompound having Formula 68 under conditions suitable for the formationof a compound having Formula 64.

[0329] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 62:

[0330] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent; each5 independently comprises a protein or peptide, for example Human serumalbumin protein, Antennapedia peptide, Kaposi fibroblast growth factorpeptide, Caiman crocodylus Ig(5) light chain peptide, HIV envelopeglycoprotein gp41 peptide, HIV-1 Tat peptide, Influenza hemagglutininenvelope glycoprotein peptide, or transportan A peptide; each R1, R2,and R3 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and each nis independently an integer from about 1 to about 10, comprising: (a)introducing a compound having Formula 93:

[0331] wherein V and n are as defined in Formula 62, to a compoundhaving Formula 86:

[0332] wherein X, W, R1, R2, R3, and n are as defined in Formula 62,under conditions suitable for the formation of a compound having Formula62.

[0333] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 63:

[0334] wherein X comprises a biologically active molecule; W comprises alinker molecule or chemical linkage that can be present or absent; Vcomprises a protein or peptide, for example Human serum albumin protein,Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caimancrocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each R1, R2, and R3independently comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, R4represents an ester, amide, or protecting group, and each n isindependently an integer from about 1 to about 10, comprising: (a)introducing a compound having Formula 96:

[0335] wherein V and R4 are as defined in Formula 63, to a compoundhaving Formula 86:

[0336] wherein X, W, R1, R2, R3, and n are as defined in Formula 63,under conditions suitable for the formation of a compound having Formula63.

[0337] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 87:

[0338] wherein X comprises a protein, peptide, antibody, lipid,phospholipid, oligosaccharide, label, biologically active molecule, forexample a vitamin such as folate, vitamin A, E, B6, B12, coenzyme,antibiotic, antiviral, nucleic acid, nucleotide, nucleoside, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, siNA, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, or polymers such as polyethylene glycol; W comprises alinker molecule or chemical linkage that can be present or absent; and Ycomprises a biologically active molecule, for example an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, peptide, protein, orantibody; R1 comprises H, alkyl, or substituted alkyl, comprising (a)coupling a compound having Formula 89:

[0339] wherein Y, W and R are as defined in Formula 87, with a compoundhaving Formula 90:

H₂N—O—X  90

[0340] wherein X is as defined in Formula 87, under conditions suitablefor the formation of a compound having Formula 87, for example bypost-synthetic conjugation of a compound having Formula 89 with acompound having Formula 90, wherein X of compound 90 comprises anenzymatic nucleic acid molecule and Y of Formula 89 comprises a peptide.

[0341] In another embodiment, the invention features a method for thesynthesis of a compound having Formula 88:

[0342] wherein X comprises a protein, peptide, antibody, lipid,phospholipid, oligosaccharide, label, biologically active molecule, forexample a vitamin such as folate, vitamin A, E, B6, B12, coenzyme,antibiotic, antiviral, nucleic acid, nucleotide, nucleoside, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, siNA, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, or polymers such as polyethylene glycol; W comprises alinker molecule or chemical linkage that can be present or absent, and Ycomprises a biologically active molecule, for example an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, peptide, protein, orantibody, comprising (a) coupling a compound having Formula 91:

[0343] wherein Y and W are as defined in Formula 88, with a compoundhaving Formula 90:

H₂N—O—X  90

[0344] wherein X is as defined in Formula 88, under conditions suitablefor the formation of a compound having Formula 88, for example bypost-synthetic conjugation of a compound having Formula 91 with acompound having Formula 90, wherein X of compound 90 comprises anenzymatic nucleic acid molecule and Y of Formula 91 comprises a peptide.

[0345] In one embodiment, the invention features a compound havingFormula 94,

X—Y—W—Y-Z  94

[0346] wherein X comprises a protein, peptide, antibody, lipid,phospholipid, oligosaccharide, label, biologically active molecule, forexample a vitamin such as folate, vitamin A, E, B6, B12, coenzyme,antibiotic, antiviral, nucleic acid, nucleotide, nucleoside, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, siNA, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, or polymers such as polyethylene glycol; each Yindependently comprises a linker or chemical linkage that can be presentor absent, W comprises a biodegradable nucleic acid linker molecule, andZ comprises a biologically active molecule, for example an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, peptide, protein, orantibody.

[0347] In another embodiment, W of a compound having Formula 94 of theinvention comprises5′-cytidine-deoxythymidine-3′,5′-deoxythymidine-cytidine-3′,5′-cytidine-deoxyuridine-3′,5′-deoxyuridine-cytidine-3′,5′-uridine-deoxythymidine-3′,or 5′-deoxythymidine-uridine-3′.

[0348] In yet another embodiment, W of a compound having Formula 94 ofthe invention comprises5′-adenosine-deoxythymidine-3′,5′-deoxythymidine-adenosine-3′,5′-adenosine-deoxyuridine-3′,or 5′-deoxyuridine-adenosine-3′.

[0349] In another embodiment, Y of a compound having Formula 94 of theinvention comprises a phosphorus containing linkage, phoshoramidatelinkage, phosphodiester linkage, phosphorothioate linkage, amidelinkage, ester linkage, carbamate linkage, disulfide linkage, oximelinkage, or morpholino linkage.

[0350] In another embodiment, compounds having Formula 89 and 91 of theinvention are synthesized by periodate oxidation of an N-terminal Serineor Threonine residue of a peptide or protein.

[0351] In one embodiment, X of compounds having Formulae 43, 44, 46-52,58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119,or 121 of the invention comprises a siNA molecule or a portion thereof.In one embodiment, the siNA molecule can be conjugated at the 5′ end,3′-end, or both 5′ and 3′ ends of the sense strand or region of thesiNA. In one embodiment, the siNA molecule can be conjugated at the3′-end of the antisense strand or region of the siNA with a compound ofthe invention. In one embodiment, both the sense strand and antisensestrands or regions of the siNA molecule are conjugated with a compoundof the invention. In one embodiment, only the sense strand or region ofthe siNA is conjugated with a compound of the invention. In oneembodiment, only the antisense strand or region of the siNA isconjugated with a compound of the invention.

[0352] In one embodiment, X of compounds having Formulae 43, 44, 46-52,58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119,or 121 of the invention comprises an enzymatic nucleic acid.

[0353] In another embodiment, X of compounds having Formulae 43, 44,46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115,118, 119, or 121 of the invention comprises an antibody. In yet anotherembodiment, X of compounds having Formulae 43, 44, 46-52, 58, 61-65,85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119, or 121 ofthe invention comprises an interferon.

[0354] In another embodiment, X of compounds having Formulae 43, 44,46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115,118, 119, or 121 of the invention comprises an antisense nucleic acid,dsRNA, ssRNA, decoy, triplex oligonucleotide, aptamer, or 2,5-A chimera.

[0355] In one embodiment, W and/or Y of compounds having Formulae 43,44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111,114, 115, 118, 119, or 121 of the invention comprises a degradable orcleavable linker, for example a nucleic acid sequence comprisingribonucleotides and/or deoxynucleotides, such as a dimer, trimer, ortetramer. A non limiting example of a nucleic acid cleavable linker isan adenosine-deoxythymidine (A-dT) dimer or a cytidine-deoxythymidine(C-dT) dimer. In yet another embodiment, W and/or V of compounds havingFormulae 43, 44, 48-51, 58, 63-65, 96, 99, 100, 107, 108, 111, 114, 115,118, 119, or 121 of the invention comprises a N-hydroxy succinimide(NHS) ester linkage, oxime linkage, disulfide linkage, phosphoramidate,phosphorothioate, phosphorodithioate, phosphodiester linkage, or NHC(O),CH₃NC(O), CONH, C(O)NCH₃, S, SO, SO₂, O, NH, NCH₃ group. In anotherembodiment, the degradable linker, W and/or Y, of compounds havingFormulae Formulae 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100,101, 107, 108, 111, 114, 115, 118, 119, or 121 of the inventioncomprises a linker that is susceptible to cleavage by carboxypeptidaseactivity.

[0356] In another embodiment, W and/or Y of Formulae Formulae 43, 44,46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114,115, 118, 119, or 121 comprises a polyethylene glycol linker havingFormula 45:

[0357] wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; and n is an integerfrom about 1 to about 100.

[0358] In one embodiment, the nucleic acid conjugates of the instantinvention are assembled by solid phase synthesis, for example on anautomated peptide synthesizer, for example a Miligen 9050 synthesizerand/or an automated oligonucleotide synthesizer such as an ABI 394,390Z, or Pharmacia OligoProcess, OligoPilot, OligoMax, or AKTAsynthesizer. In another embodiment, the nucleic acid conjugates of theinvention are assembled post synthetically, for example, following solidphase oligonucleotide synthesis (see for example FIG. 15).

[0359] In another embodiment, V of compounds having Formula 58-63 and 96comprise peptides having SEQ ID NOS: 14-23 (Table III).

[0360] In one embodiment, the nucleic acid conjugates of the instantinvention are assembled post synthetically, for example, following solidphase oligonucleotide synthesis.

[0361] The present invention provides compositions and conjugatescomprising nucleosidic and non-nucleosidic derivatives. The presentinvention also provides nucleic acid, polynucleotide and oligonucleotidederivatives including RNA, DNA, and PNA based conjugates. The attachmentof compounds of the invention to nucleosides, nucleotides,non-nucleosides, and nucleic acid molecules is provided at any positionwithin the molecule, for example, at internucleotide linkages,nucleosidic sugar hydroxyl groups such as 5′, 3′, and 2′-hydroxyls,and/or at nucleobase positions such as amino and carbonyl groups.

[0362] The exemplary conjugates of the invention are described ascompounds of the formulae herein, however, other peptide, protein,phospholipid, and poly-alkyl glycol derivatives are provided by theinvention, including various analogs of the compounds of formulae 1-122,including but not limited to different isomers of the compoundsdescribed herein.

[0363] In one embodiment, the present invention features molecules,compositions and conjugates of molecules, for example, non-nucleosidicsmall molecules, nucleosides, nucleotides, and nucleic acids, such asenzymatic nucleic acid molecules, antisense nucleic acids, 2-5Aantisense chimeras, triplex oligonucleotides, decoys, siNA, allozymes,aptamers, and antisense nucleic acids containing RNA cleaving chemicalgroups.

[0364] The exemplary folate conjugates of the invention are described ascompounds shown by formulae herein, however, other folate and antifolatederivatives are provided by the invention, including various folateanalogs of the formulae of the invention, including dihydrofloates,tetrahydrofolates, tetrahydorpterins, folinic acid, pteropolyglutamicacid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroicacids. As used herein, the term “folate” is meant to refer to folate andfolate derivatives, including pteroic acid derivatives and analogs.

[0365] The present invention features compositions and conjugates tofacilitate delivery of molecules into a biological system such as cells.The conjugates provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes. The present invention encompasses the design and synthesis ofnovel agents for the delivery of molecules, including but not limited tosmall molecules, lipids, nucleosides, nucleotides, nucleic acids,negatively charged polymers and other polymers, for example proteins,peptides, carbohydrates, or polyamines. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system. The compounds of the invention generally shownin Formulae herein are expected to improve delivery of molecules into anumber of cell types originating from different tissues, in the presenceor absence of serum.

[0366] In another embodiment, the present invention features methods tomodulate gene expression, for example, genes involved in the progressionand/or maintenance of cancer or in a viral infection. For example, inone embodiment, the invention features the use of one or more of thenucleic acid-based molecules and methods independently or in combinationto inhibit the expression of the gene(s) encoding proteins associatedwith cancerous conditions, for example breast cancer, lung cancer,colorectal cancer, brain cancer, esophageal cancer, stomach cancer,bladder cancer, pancreatic cancer, cervical cancer, head and neckcancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrugresistant cancer associated genes.

[0367] In another embodiment, the invention features the use of one ormore of the nucleic acid-based molecules and methods independently or incombination to inhibit the expression of the gene(s) encoding viralproteins, for example HIV, HBV, HCV, CMV, RSV, HSV, poliovirus,influenza, rhinovirus, west nile virus, Ebola virus, foot and mouthvirus, and papilloma virus associated genes.

[0368] In one embodiment, the invention features the use of an enzymaticnucleic acid molecule conjugate comprising compounds of formulae 1-122,preferably in the hammerhead, NCH, G-cleaver, amberzyme, zinzyme and/orDNAzyme motif, to inhibit the expression of cancer and virus associatedgenes.

[0369] In another embodiment, the invention features the use of anenzymatic nucleic acid molecule as a conjugate. These enzymatic nucleicacids can catalyze the hydrolysis of RNA phosphodiester bonds in trans(and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseenzymatic nucleic acids. Without being bound by any particular theory,in general, enzymatic nucleic acids act by first binding to a targetRNA. Such binding occurs through the target binding portion of anenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA destroys its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets. Thus, a single enzymaticnucleic acid molecule is able to cleave many molecules of target RNA. Inaddition, the enzymatic nucleic acid is a highly specific inhibitor ofgene expression, with the specificity of inhibition depending not onlyon the base-pairing mechanism of binding to the target RNA, but also onthe mechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of an enzymatic nucleic acid.

[0370] In one embodiment of the invention described herein, theenzymatic nucleic acid molecule component of the conjugate is formed ina hammerhead or hairpin motif, but can also be formed in the motif of ahepatitis delta virus, group I intron, group II intron or RNase P RNA(in association with an RNA guide sequence), Neurospora VS RNA,DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of suchhammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992,AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampelet al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929,Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene,82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowrira &McSwiggen, U.S. Pat. No. 5,631,359; of the hepatitis delta virus motifis described by Perrotta and Been, 1992 Biochemistry 31, 16; of theRNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster andAltman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res.24, 835; Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363);Group II introns are described by Griffin et al., 1995, Chem. Biol. 2,761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al.,International PCT Publication No. WO 96/22689; of the Group I intron byCech et al., U.S. Pat. No. 4,987,071 and of DNAzymes by Usman et al.,International PCT Publication No. WO 95/11304; Chartrand et al., 1995,NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al.,1997, PNAS 94, 4262, and Beigelman et al., International PCT publicationNo. WO 99/55857. NCH cleaving motifs are described in Ludwig & Sproat,International PCT Publication No. WO 98/58058; and G-cleavers aredescribed in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 andEckstein et al., International PCT Publication No. WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993),Amberzyme (Class I motif; FIG. 3; Beigelman et al., U.S. Ser. No.09/301,511) and Zinzyme (FIG. 4) (Beigelman et al., U.S. Ser. No.09/301,511), all incorporated by reference herein including drawings,can also be used in the present invention. These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule (Cech et al.,U.S. Pat. No. 4,987,071).

[0371] In one embodiment of the present invention, a nucleic acidmolecule component of a conjugate of the instant invention can be about12 to about 100 nucleotides in length. For example, enzymatic nucleicacid molecules of the invention are preferably about 15 to about 50nucleotides in length, more preferably about 25 to about 40 nucleotidesin length, e.g., 34, 36, or 38 nucleotides in length (for example seeJarvis et al., 1996, J., Biol. Chem., 271, 29107-29112). ExemplaryDNAzymes of the invention are preferably about 15 to about 40nucleotides in length, more preferably about 25 to about 35 nucleotidesin length, e.g., 29, 30, 31, or 32 nucleotides in length (see forexample Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrandet al., 1995, Nucleic Acids Research, 23, 4092-4096). Exemplaryantisense molecules of the invention are preferably about 15 to about 75nucleotides in length, more preferably about 20 to about 35 nucleotidesin length, e.g., 25, 26, 27, or 28 nucleotides in length (see, forexample, Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997,Nature Biotechnology, 15, 537-541). Exemplary triplex formingoligonucleotide molecules of the invention are preferably about 10 toabout 40 nucleotides in length, more preferably about 12 to about 25nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length(see for example Maher et al., 1990, Biochemistry, 29, 8820-8826;Strobel and Dervan, 1990, Science, 249, 73-75). Exemplary doublestranded siNA molecules of the invention comprise about 19 to about 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, 24, or 25nucleotides in length, for each strand of the siNA molecule. Exemplarysingle stranded siNA molecules of the invention are about 38 to about 50nucleotides in length, e.g., about 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 nucleotides in length. The length of the nucleic acidmolecules described and exemplified herein are not limiting within thegeneral size ranges stated.

[0372] The conjugates of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The conjugates and/or conjugatecomplexes can be locally administered to relevant tissues ex vivo, or invivo through injection or infusion pump, with or without theirincorporation in biopolymers. The compositions and conjugates of theinstant invention, individually, or in combination or in conjunctionwith other drugs, can be used to treat diseases or conditions discussedabove. For example, to treat a disease or condition associated with thelevels of a pathogenic protein, the patient can be treated, or otherappropriate cells can be treated, as is evident to those skilled in theart, individually or in combination with one or more drugs underconditions suitable for the treatment.

[0373] In a further embodiment, the described molecules can be used incombination with other known treatments to treat conditions or diseasesdiscussed above. For example, the described molecules can be used incombination with one or more known therapeutic agents to treat breast,lung, prostate, colorectal, brain, esophageal, bladder, pancreatic,cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma,multidrug resistant cancers, and/or HIV, HBV, HCV, CMV, RSV, HSV,poliovirus, influenza, rhinovirus, west nile virus, Ebola virus, footand mouth virus, and papilloma virus infection.

[0374] Included in another embodiment are a series of multi-domaincellular transport vehicles (MCTV) including one or more compounds ofFormulae 1-122 herein that enhance the cellular uptake and transmembranepermeability of negatively charged molecules in a variety of cell types.The compounds of the invention are used either alone or in combinationwith other compounds with a neutral or a negative charge including butnot limited to neutral lipid and/or targeting components, to improve theeffectiveness of the formulation or conjugate in delivering andtargeting the predetermined compound or molecule to cells. Anotherembodiment of the invention encompasses the utility of these compoundsfor increasing the transport of other impermeable and/or lipophiliccompounds into cells. Targeting components include ligands for cellsurface receptors including, peptides and proteins, glycolipids, lipids,carbohydrates, and their synthetic variants, for example folatereceptors.

[0375] In another embodiment, the compounds of the invention areprovided as a surface component of a lipid aggregate, such as a liposomeencapsulated with the predetermined molecule to be delivered. Liposomes,which can be unilamellar or multilamellar, can introduce encapsulatedmaterial into a cell by different mechanisms. For example, the liposomecan directly introduce its encapsulated material into the cell cytoplasmby fusing with the cell membrane. Alternatively, the liposome can becompartmentalized into an acidic vacuole (i.e., an endosome) and itscontents released from the liposome and out of the acidic vacuole intothe cellular cytoplasm.

[0376] In one embodiment the invention features a lipid aggregateformulation of the compounds described herein, includingphosphatidylcholine (of varying chain length; e.g., egg yolkphosphatidylcholine), cholesterol, a cationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000(DSPE-PEG2000). The cationic lipid component of this lipid aggregate canbe any cationic lipid known in the art such as dioleoyl1,2-diacyl-3-trimethylammonium-propane (DOTAP). In another embodimentthis cationic lipid aggregate comprises a covalently bound compounddescribed in any of the Formulae herein.

[0377] In another embodiment, polyethylene glycol (PEG) is covalentlyattached to the compounds of the present invention. The attached PEG canbe any molecular weight but is preferably between 2000-50,000 daltons.

[0378] The compounds and methods of the present invention are useful forintroducing nucleotides, nucleosides, nucleic acid molecules, lipids,peptides, proteins, and/or non-nucleosidic small molecules into a cell.For example, the invention can be used for nucleotide, nucleoside,nucleic acid, lipids, peptides, proteins, and/or non-nucleosidic smallmolecule delivery where the corresponding target site of action existsintracellularly.

[0379] In one embodiment, the compounds of the instant invention provideconjugates of molecules that can interact with cellular receptors, suchas high affinity folate receptors and ASGPr receptors, and provide anumber of features that allow the efficient delivery and subsequentrelease of conjugated compounds across biological membranes. Thecompounds utilize chemical linkages between the receptor ligand and thecompound to be delivered of length that can interact preferentially withcellular receptors. Furthermore, the chemical linkages between theligand and the compound to be delivered can be designed as degradablelinkages, for example by utilizing a phosphate linkage that is proximalto a nucleophile, such as a hydroxyl group. Deprotonation of thehydroxyl group or an equivalent group, as a result of pH or interactionwith a nuclease, can result in nucleophilic attack of the phosphateresulting in a cyclic phosphate intermediate that can be hydrolyzed.This cleavage mechanism is analogous RNA cleavage in the presence of abase or RNA nuclease. Alternately, other degradable linkages can beselected that respond to various factors such as UV irradiation,cellular nucleases, pH, temperature etc. The use of degradable linkagesallows the delivered compound to be released in a predetermined system,for example in the cytoplasm of a cell, or in a particular cellularorganelle.

[0380] The present invention also provides ligand derivedphosphoramidites that are readily conjugated to compounds and moleculesof interest. Phosphoramidite compounds of the invention permit thedirect attachment of conjugates to molecules of interest without theneed for using nucleic acid phosphoramidite species as scaffolds. Assuch, the used of phosphoramidite chemistry can be used directly incoupling the compounds of the invention to a compound of interest,without the need for other condensation reactions, such as condensationof the ligand to an amino group on the nucleic acid, for example at theN6 position of adenosine or a 2′-deoxy-2′-amino function. Additionally,compounds of the invention can be used to introduce non-nucleic acidbased conjugated linkages into oligonucleotides that can provide moreefficient coupling during oligonucleotide synthesis than the use ofnucleic acid-based phosphoramidites. This improved coupling can takeinto account improved steric considerations of abasic or non-nucleosidicscaffolds bearing pendant alkyl linkages.

[0381] Compounds of the invention utilizing triphosphate groups can beutilized in the enzymatic incorporation of conjugate molecules intooligonucleotides. Such enzymatic incorporation is useful when conjugatesare used in post-synthetic enzymatic conjugation or selection reactions,(see for example Matulic-Adamic et al., 2000, Bioorg. Med. Chem. Lett.,10, 1299-1302; Lee et al., 2001, NAR., 29, 1565-1573; Joyce, 1989, Gene,82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992,Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268;Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17,89-93; Kumar et al., 1995, FASEB J, 9, 1183; Breaker, 1996, Curr. Op.Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra;Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish etal., 1997, Biochemistry 36, 6495; Kuwabara et al., 2000, Curr. Opin.Chem. Biol., 4, 669).

[0382] Compounds of the invention can be used to detect the presence ofa target molecule in a biological system, such as tissue, cell or celllysate. Examples of target molecules include nucleic acids, proteins,peptides, antibodies, polysaccharides, lipids, hormones, sugars, metals,microbial or cellular metabolites, analytes, pharmaceuticals, and otherorganic and inorganic molecules or other biomolecules in a sample. Thecompounds of the instant invention can be conjugated to a predeterminedcompound or molecule that is capable of interacting with the targetmolecule in the system and providing a detectable signal or response.Various compounds and molecules known in the art that can be used inthese applications include but are not limited to antibodies, labeledantibodies, allozymes, aptamers, labeled nucleic acid probes, molecularbeacons, fluorescent molecules, radioisotopes, polysaccharides, and anyother compound capable of interacting with the target molecule andgenerating a detectable signal upon target interaction. For example,such compounds are described in Application entitled “NUCLEIC ACIDSENSOR MOLECULES”, U.S. Ser. No. 09/800,594 filed on Mar. 6, 2001 (Notyet assigned; Attorney Docket No. MBHB00-816-A 700.001) with inventorsNassim Usman and James A. McSwiggen, which is incorporated by referencein its entirety, including the drawings.

[0383] The term “biodegradable nucleic acid linker molecule” as usedherein, refers to a nucleic acid molecule that is designed as abiodegradable linker to connect one molecule to another molecule, forexample, a biologically active molecule. The stability of thebiodegradable nucleic acid linker molecule can be modulated by usingvarious combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, for example 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus based linkage, forexample a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

[0384] The term “biodegradable” as used herein, refers to degradation ina biological system, for example enzymatic degradation or chemicaldegradation.

[0385] The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive molecules contemplated by the instant invention includetherapeutically active molecules such as antibodies, hormones,antivirals, peptides, proteins, chemotherapeutics, small molecules,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

[0386] The term “phospholipid” as used herein, refers to a hydrophobicmolecule comprising at least one phosphorus group. For example, aphospholipid can comprise a phosphorus containing group and saturated orunsaturated alkyl group, optionally substituted with OH, COOH, oxo,amine, or substituted or unsubstituted aryl groups.

[0387] The term “nitrogen containing group” as used herein refers to anychemical group or moiety comprising a nitrogen or substituted nitrogen.Non-limiting examples of nitrogen containing groups include amines,substituted amines, amides, alkylamines, amino acids such as arginine orlysine, polyamines such as spermine or spermidine, cyclic amines such aspyridines, pyrimidines including uracil, thymine, and cytosine,morpholines, phthalimides, and heterocyclic amines such as purines,including guanine and adenine.

[0388] The term “target molecule” as used herein, refers to nucleic acidmolecules, proteins, peptides, antibodies, polysaccharides, lipids,sugars, metals, microbial or cellular metabolites, analytes,pharmaceuticals, and other organic and inorganic molecules that arepresent in a system.

[0389] By “inhibit” or “down-regulate” it is meant that the expressionof the gene, or level of RNAs or equivalent RNAs encoding one or moreprotein subunits, or activity of one or more protein subunits, such aspathogenic protein, viral protein or cancer related protein subunit(s),is reduced below that observed in the absence of the compounds orcombination of compounds of the invention. In one embodiment, inhibitionor down-regulation with an enzymatic nucleic acid molecule preferably isbelow that level observed in the presence of an enzymatically inactiveor attenuated molecule that is able to bind to the same site on thetarget RNA, but is unable to cleave that RNA. In another embodiment,inhibition or down-regulation with antisense oligonucleotides ispreferably below that level observed in the presence of, for example, anoligonucleotide with scrambled sequence or with mismatches. In anotherembodiment, inhibition or down-regulation of viral or oncogenic RNA,protein, or protein subunits with a compound of the instant invention isgreater in the presence of the compound than in its absence.

[0390] By “up-regulate” is meant that the expression of the gene, orlevel of RNAs or equivalent RNAs encoding one or more protein subunits,or activity of one or more protein subunits, such as viral or oncogenicprotein subunit(s), is greater than that observed in the absence of thecompounds or combination of compounds of the invention. For example, theexpression of a gene, such as a viral or cancer related gene, can beincreased in order to treat, prevent, ameliorate, or modulate apathological condition caused or exacerbated by an absence or low levelof gene expression.

[0391] By “modulate” is meant that the expression of the gene, or levelof RNAs or equivalent RNAs encoding one or more protein subunits, oractivity of one or more protein subunit(s) of a protein, for example aviral or cancer related protein is upregulated or down-regulated, suchthat the expression, level, or activity is greater than or less thanthat observed in the absence of the compounds or combination ofcompounds of the invention.

[0392] The term “enzymatic nucleic acid molecule” as used herein refersto a nucleic acid molecule which has complementarity in a substratebinding region to a specified gene target, and also has an enzymaticactivity which is active to specifically cleave target RNA. That is, theenzymatic nucleic acid molecule is able to intermolecularly cleave RNAand thereby inactivate a target RNA molecule. These complementaryregions allow sufficient hybridization of the enzymatic nucleic acidmolecule to the target RNA and thus permit cleavage. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% canalso be useful in this invention (see for example Werner and Uhlenbeck,1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids canbe modified at the base, sugar, and/or phosphate groups. The termenzymatic nucleic acid is used interchangeably with phrases such asribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme oraptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The specific enzymatic nucleic acid molecules described in the instantapplication are not limiting in the invention and those skilled in theart will recognize that all that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target nucleicacid regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart a nucleic acidcleaving and/or ligation activity to the molecule (Cech et al., U.S.Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).

[0393] The term “nucleic acid molecule” as used herein, refers to amolecule having nucleotides. The nucleic acid can be single, double, ormultiple stranded and can comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof.

[0394] The term “enzymatic portion” or “catalytic domain” as used hereinrefers to that portion/region of the enzymatic nucleic acid moleculeessential for cleavage of a nucleic acid substrate (for example see FIG.1).

[0395] The term “substrate binding arm” or “substrate binding domain” asused herein refers to that portion/region of a enzymatic nucleic acidwhich is able to interact, for example via complementarity (i.e., ableto base-pair with), with a portion of its substrate. Preferably, suchcomplementarity is 100%, but can be less if desired. For example, as fewas 10 bases out of 14 can be base-paired (see for example Werner andUhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al.,1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of sucharms are shown generally in FIGS. 1-4. That is, these arms containsequences within a enzymatic nucleic acid which are intended to bringenzymatic nucleic acid and target RNA together through complementarybase-pairing interactions. The enzymatic nucleic acid of the inventioncan have binding arms that are contiguous or non-contiguous and can beof varying lengths. The length of the binding arm(s) are preferablygreater than or equal to four nucleotides and of sufficient length tostably interact with the target RNA; preferably 12-100 nucleotides; morepreferably 14-24 nucleotides long (see for example Werner and Uhlenbeck,supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranceet al., 1993, EMBO J., 12, 2567-73). If two binding arms are chosen, thedesign is such that the length of the binding arms are symmetrical(i.e., each of the binding arms is of the same length; e.g., five andfive nucleotides, or six and six nucleotides, or seven and sevennucleotides long) or asymmetrical (i.e., the binding arms are ofdifferent length; e.g., six and three nucleotides; three and sixnucleotides long; four and five nucleotides long; four and sixnucleotides long; four and seven nucleotides long; and the like).

[0396] The term “Inozyme” or “NCH” motif as used herein, refers to anenzymatic nucleic acid molecule comprising a motif as is generallydescribed as NCH Rz in FIG. 1. Inozymes possess endonuclease activity tocleave RNA substrates having a cleavage triplet NCH/, where N is anucleotide, C is cytidine and H is adenosine, uridine or cytidine, and /represents the cleavage site. H is used interchangeably with X. Inozymescan also possess endonuclease activity to cleave RNA substrates having acleavage triplet NCN/, where N is a nucleotide, C is cytidine, and /represents the cleavage site. “I” in FIG. 2 represents an Inosinenucleotide, preferably a ribo-Inosine or xylo-Inosine nucleoside.

[0397] The term “G-cleaver” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described asG-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity tocleave RNA substrates having a cleavage triplet NYN/, where N is anucleotide, Y is uridine or cytidine and / represents the cleavage site.G-cleavers can be chemically modified as is generally shown in FIG. 2.

[0398] The term “amberzyme” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described inFIG. 2. Amberzymes possess endonuclease activity to cleave RNAsubstrates having a cleavage triplet NG/N, where N is a nucleotide, G isguanosine, and / represents the cleavage site. Amberzymes can bechemically modified to increase nuclease stability through substitutionsas are generally shown in FIG. 3. In addition, differing nucleosideand/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′loops shown in the figure. Amberzymes represent a non-limiting exampleof an enzymatic nucleic acid molecule that does not require aribonucleotide (2′-OH) group within its own nucleic acid sequence foractivity.

[0399] The term “zinzyme” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described inFIG. 3. Zinzymes possess endonuclease activity to cleave RNA substrateshaving a cleavage triplet including but not limited to YG/Y, where Y isuridine or cytidine, and G is guanosine and / represents the cleavagesite. Zinzymes can be chemically modified to increase nuclease stabilitythrough substitutions as are generally shown in FIG. 3, includingsubstituting 2′-O-methyl guanosine nucleotides for guanosinenucleotides. In addition, differing nucleotide and/or non-nucleotidelinkers can be used to substitute the 5′-gaaa-2′ loop shown in thefigure. Zinzymes represent a non-limiting example of an enzymaticnucleic acid molecule that does not require a ribonucleotide (2′-OH)group within its own nucleic acid sequence for activity.

[0400] The term ‘DNAzyme’ as used herein, refers to an enzymatic nucleicacid molecule that does not require the presence of a 2′-OH group forits activity. In particular embodiments the enzymatic nucleic acidmolecule can have an attached linker(s) or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. DNAzymes can be synthesized chemically or expressedendogenously in vivo, by means of a single stranded DNA vector orequivalent thereof. An example of a DNAzyme is shown in FIG. 4 and isgenerally reviewed in Usman et al., International PCT Publication No. WO95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995,Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999,Nature Biotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am.Chem. Soc., 122, 2433-39. Additional DNAzyme motifs can be selected forusing techniques similar to those described in these references, andhence, are within the scope of the present invention.

[0401] The term “sufficient length” as used herein, refers to anoligonucleotide of length great enough to provide the intended functionunder the expected condition, i.e., greater than or equal to 3nucleotides. For example, for binding arms of enzymatic nucleic acid“sufficient length” means that the binding arm sequence is long enoughto provide stable binding to a target site under the expected bindingconditions. Preferably, the binding arms are not so long as to preventuseful turnover of the nucleic acid molecule.

[0402] The term “stably interact” as used herein, refers to interactionof the oligonucleotides with target nucleic acid (e.g., by forminghydrogen bonds with complementary nucleotides in the target underphysiological conditions) that is sufficient to the intended purpose(e.g., cleavage of target RNA by an enzyme).

[0403] The term “homology” as used herein, refers to the nucleotidesequence of two or more nucleic acid molecules is partially orcompletely identical.

[0404] The term “antisense nucleic acid”, as used herein, refers to anon-enzymatic nucleic acid molecule that binds to target RNA by means ofRNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993Nature 365, 566) interactions and alters the activity of the target RNA(for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf etal., U.S. Pat. No. 5,849,902). Typically, antisense molecules arecomplementary to a target sequence along a single contiguous sequence ofthe antisense molecule. However, in certain embodiments, an antisensemolecule can bind to substrate such that the substrate molecule forms aloop, and/or an antisense molecule can bind such that the antisensemolecule forms a loop. Thus, the antisense molecule can be complementaryto two (or even more) non-contiguous substrate sequences or two (or evenmore) non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. For a review of currentantisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274,21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al.,1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol.,313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke,1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be usedto target RNA by means of DNA-RNA interactions, thereby activating RNaseH, which digests the target RNA in the duplex. The antisenseoligonucleotides can comprise one or more RNAse H activating region,which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof.

[0405] The term “RNase H activating region” as used herein, refers to aregion (generally greater than or equal to 4-25 nucleotides in length,preferably from 5-11 nucleotides in length) of a nucleic acid moleculecapable of binding to a target RNA to form a non-covalent complex thatis recognized by cellular RNase H enzyme (see for example Arrow et al.,U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). TheRNase H enzyme binds to the nucleic acid molecule-target RNA complex andcleaves the target RNA sequence. The RNase H activating regioncomprises, for example, phosphodiester, phosphorothioate (preferably atleast four of the nucleotides are phosphorothiote substitutions; morespecifically, 4-11 of the nucleotides are phosphorothiotesubstitutions); phosphorodithioate, 5′-thiophosphate, ormethylphosphonate backbone chemistry or a combination thereof. Inaddition to one or more backbone chemistries described above, the RNaseH activating region can also comprise a variety of sugar chemistries.For example, the RNase H activating region can comprise deoxyribose,arabino, fluoroarabino or a combination thereof, nucleotide sugarchemistry. Those skilled in the art will recognize that the foregoingare non-limiting examples and that any combination of phosphate, sugarand base chemistry of a nucleic acid that supports the activity of RNaseH enzyme is within the scope of the definition of the RNase H activatingregion and the instant invention.

[0406] The term “2-5A antisense chimera” as used herein, refers to anantisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linkedadenylate residue. These chimeras bind to target RNA in asequence-specific manner and activate a cellular 2-5A-dependentribonuclease which, in turn, cleaves the target RNA (Torrence et al.,1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000,Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol.Ther., 78, 55-113).

[0407] The term “triplex forming oligonucleotides” as used herein,refers to an oligonucleotide that can bind to a double-stranded DNA in asequence-specific manner to form a triple-strand helix. Formation ofsuch triple helix structure has been shown to inhibit transcription ofthe targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci.USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al.,2000, Biochim. Biophys. Acta, 1489, 181-206).

[0408] The term “gene” it as used herein, refers to a nucleic acid thatencodes an RNA, for example, nucleic acid sequences including but notlimited to structural genes encoding a polypeptide.

[0409] The term “pathogenic protein” as used herein, refers toendogenous or exongenous proteins that are associated with a diseasestate or condition, for example a particular cancer or viral infection.

[0410] The term “complementarity” refers to the ability of a nucleicacid to form hydrogen bond(s) with another RNA sequence by eithertraditional Watson-Crick or other non-traditional types. In reference tothe nucleic molecules of the present invention, the binding free energyfor a nucleic acid molecule with its target or complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., enzymatic nucleic acid cleavage, antisense or triplehelix inhibition. Determination of binding free energies for nucleicacid molecules is well known in the art (see, e.g., Turner et al., 1987,CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat.Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule which can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%,90%, and 100% complementary). “Perfectly complementary” means that allthe contiguous residues of a nucleic acid sequence will hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence.

[0411] The term “RNA” as used herein, refers to a molecule comprising atleast one ribonucleotide residue. By “ribonucleotide” or “2′-OH” ismeant a nucleotide with a hydroxyl group at the 2′ position of aβ-D-ribo-furanose moiety.

[0412] The term “decoy RNA” as used herein, refers to a RNA molecule oraptamer that is designed to preferentially bind to a predeterminedligand. Such binding can result in the inhibition or activation of atarget molecule. The decoy RNA or aptamer can compete with a naturallyoccurring binding target for the binding of a specific ligand. Forexample, it has been shown that over-expression of HIV trans-activationresponse (TAR) RNA can act as a “decoy” and efficiently binds HIV tatprotein, thereby preventing it from binding to TAR sequences encoded inthe HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608). This is but aspecific example and those in the art will recognize that otherembodiments can be readily generated using techniques generally known inthe art, see for example Gold et al., 1995, Annu. Rev. Biochem., 64,763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin.Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann andPatel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry,45, 1628. Similarly, a decoy RNA can be designed to bind to a receptorand block the binding of an effector molecule or a decoy RNA can bedesigned to bind to receptor of interest and prevent interaction withthe receptor.

[0413] The term “single stranded RNA” (ssRNA) as used herein refers to anaturally occurring or synthetic ribonucleic acid molecule comprising alinear single strand, for example a ssRNA can be a messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA) etc. of a gene.

[0414] The term “single stranded DNA” (ssDNA) as used herein refers to anaturally occurring or synthetic deoxyribonucleic acid moleculecomprising a linear single strand, for example, a ssDNA can be a senseor antisense gene sequence or EST (Expressed Sequence Tag).

[0415] The term “double stranded RNA” or “dsRNA” as used herein refersto a double stranded RNA molecule capable of RNA interference, includingshort interfering RNA (siNA).

[0416] The term “short interfering nucleic acid”, “siNA”, “shortinterfering RNA”, “siRNA”, “short interfering nucleic acid molecule”,“short interfering oligonucleotide molecule”, or “chemically-modifiedshort interfering nucleic acid molecule” as used herein refers to anynucleic acid molecule capable of inhibiting or down regulating geneexpression or viral replication, for example by mediating RNAinterference “RNAi” or gene silencing in a sequence-specific manner; seefor example Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001,Nature, 411, 494-498; and Kreutzer et al., International PCT PublicationNo. WO 00/44895; Zemicka-Goetz et al., International PCT Publication No.WO 01/36646; Fire, International PCT Publication No. WO 99/32619;Plaetinck et al., International PCT Publication No. WO 00/01846; Melloand Fire, International PCT Publication No. WO 01/29058;Deschamps-Depaillette, International PCT Publication No. WO 99/07409;and Li et al., International PCT Publication No. WO 00/44914; Allshire,2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297,1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al.,2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297,2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002,Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297,1831). Non limiting examples of siNA molecules of the invention aredescribed in Haeberli et al., PCT/US03/05346 and McSwiggen et al.,PCT/US03/05028, both incorporated by reference herein in their entiretyincluding the drawings, and in FIGS. 34-42 herein. Chemicalmodifications described in Haeberli et al., PCT/US03/05346 and McSwiggenet al., PCT/US03/05028 and/or shown in Table IV can be applied to anysiNA sequence of the invention. For example the siNA can be adouble-stranded polynucleotide molecule comprising self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be assembled from twoseparate oligonucleotides, where one strand is the sense strand and theother is the antisense strand, wherein the antisense and sense strandsare self-complementary (i.e. each strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 19 base pairs); the antisense strand comprises nucleotide sequencethat is complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. Alternatively, the siNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha hairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiment, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering, nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), translational silencing, and others. In addition, as usedherein, the term RNAi is meant to be equivalent to other terms used todescribe sequence specific RNA interference, such as posttranscriptional gene silencing, or epigenetics. For example, siNAmolecules of the invention can be used to epigenetically silence genesat both the post-transcriptional level or the pre-transcriptional level.In a non-limiting example, epigenetic regulation of gene expression bysiNA molecules of the invention can result from siNA mediatedmodification of chromatin structure to alter gene expression (see, forexample, Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002,Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; andHall et al., 2002, Science, 297, 2232-2237).

[0417] The term “allozyme” as used herein refers to an allostericenzymatic nucleic acid molecule, see for example see for example Georgeet al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat.No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan andEllington, International PCT publication No. WO 00/24931, Breaker etal., International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., International PCT publication No. WO 99/29842.

[0418] The term “cell” as used herein, refers to its usual biologicalsense, and does not refer to an entire multicellular organism. The cellcan, for example, be in vitro, e.g., in cell culture, or present in amulticellular organism, including, e.g., birds, plants and mammals suchas humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cellcan be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalianor plant cell).

[0419] The term “highly conserved sequence region” as used herein,refers to a nucleotide sequence of one or more regions in a target genedoes not vary significantly from one generation to the other or from onebiological system to the other.

[0420] The term “non-nucleotide” as used herein, refers to any group orcompound which can be incorporated into a nucleic acid chain in theplace of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound is abasic in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine.

[0421] The term “nucleotide” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are recognized in the art to include natural bases(standard), and modified bases well known in the art. Such bases aregenerally located at the 1′ position of a nucleotide sugar moiety.Nucleotides generally comprise a base, sugar and a phosphate group. Thenucleotides can be unmodified or modified at the sugar, phosphate and/orbase moiety, (also referred to interchangeably as nucleotide analogs,modified nucleotides, non-natural nucleotides, non-standard nucleotidesand other; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; Uhlman & Peyman, supraall are hereby incorporated by reference herein). There are severalexamples of modified nucleic acid bases known in the art as summarizedby Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of chemically modified and other natural nucleicacid bases that can be introduced into nucleic acids include, forexample, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil,dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine, guanine, cytosine and uracilat 1′ position or their equivalents; such bases can be used at anyposition, for example, within the catalytic core of an enzymatic nucleicacid molecule and/or in the substrate-binding regions of the nucleicacid molecule.

[0422] The term “nucleoside” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a sugar. Nucleosides arerecognized in the art to include natural bases (standard), and modifiedbases well known in the art. Such bases are generally located at the 1′position of a nucleoside sugar moiety. Nucleosides generally comprise abase and sugar group. The nucleosides can be unmodified or modified atthe sugar, and/or base moiety, (also referred to interchangeably asnucleoside analogs, modified nucleosides, non-natural nucleosides,non-standard nucleosides and other; see for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra all are hereby incorporated by reference herein).There are several examples of modified nucleic acid bases known in theart as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.Some of the non-limiting examples of chemically modified and othernatural nucleic acid bases that can be introduced into nucleic acidsinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil,dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleoside bases other than adenine, guanine, cytosine and uracilat 1′ position or their equivalents; such bases can be used at anyposition, for example, within the catalytic core of an enzymatic nucleicacid molecule and/or in the substrate-binding regions of the nucleicacid molecule.

[0423] The term “cap structure” as used herein, refers to chemicalmodifications, which have been incorporated at either terminus of theoligonucleotide (see for example Wincott et al., WO 97/26270,incorporated by reference herein). These terminal modifications protectthe nucleic acid molecule from exonuclease degradation, and can help indelivery and/or localization within a cell. The cap can be present atthe 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can bepresent on both terminus. In non-limiting examples, the 5′-cap includesinverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

[0424] The term “abasic” as used herein, refers to sugar moietieslacking a base or having other chemical groups in place of a base at the1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasicribose derivative (for more details see Wincott et al., InternationalPCT publication No. WO 97/26270).

[0425] The term “unmodified nucleoside” as used herein, refers to one ofthe bases adenine, cytosine, guanine, thymine, uracil joined to the 1′carbon of β-D-ribo-furanose.

[0426] The term “modified nucleoside” as used herein, refers to anynucleotide base which contains a modification in the chemical structureof an unmodified nucleotide base, sugar and/or phosphate.

[0427] The term “consists essentially of” as used herein, is meant thatthe active nucleic acid molecule of the invention, for example, anenzymatic nucleic acid molecule, contains an enzymatic center or coreequivalent to those in the examples, and binding arms able to bind RNAsuch that cleavage at the target site occurs. Other sequences can bepresent which do not interfere with such cleavage. Thus, a core regioncan, for example, include one or more loop, stem-loop structure, orlinker which does not prevent enzymatic activity. For example, a coresequence for a hammerhead enzymatic nucleic acid can comprise aconserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by“X”, where X is 5′-GCCGUUAGGC-3′ (SEQ ID NO 1), or any other Stem IIregion known in the art, or a nucleotide and/or non-nucleotide linker.Similarly, for other nucleic acid molecules of the instant invention,such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5Aantisense, triplex forming nucleic acid, and decoy nucleic acids, othersequences or non-nucleotide linkers can be present that do not interferewith the function of the nucleic acid molecule.

[0428] Sequence X can be a linker of ≧2 nucleotides in length,preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where thenucleotides can preferably be internally base-paired to form a stem ofpreferably ≧2 base pairs. In yet another embodiment, the nucleotidelinker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Revaptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Goldet al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington,1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSHLaboratory Press). A “nucleic acid aptamer” as used herein is meant toindicate a nucleic acid sequence capable of interacting with a ligand.The ligand can be any natural or a synthetic molecule, including but notlimited to a resin, metabolites, nucleosides, nucleotides, drugs,toxins, transition state analogs, peptides, lipids, proteins, aminoacids, nucleic acid molecules, hormones, carbohydrates, receptors,cells, viruses, bacteria and others.

[0429] Alternatively or in addition, sequence X can be a non-nucleotidelinker. Non-nucleotides can include abasic nucleotide, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarboncompounds. Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound which can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment,the invention features an enzymatic nucleic acid molecule having one ormore non-nucleotide moieties, and having enzymatic activity to cleave anRNA or DNA molecule.

[0430] The term “patient” as used herein, refers to an organism, whichis a donor or recipient of explanted cells or the cells themselves.“Patient” also refers to an organism to which the nucleic acid moleculesof the invention can be administered. Preferably, a patient is a mammalor mammalian cells. More preferably, a patient is a human or humancells.

[0431] The term “enhanced enzymatic activity” as used herein, includesactivity measured in cells and/or in vivo where the activity is areflection of both the catalytic activity and the stability of thenucleic acid molecules of the invention. In this invention, the productof these properties can be increased in vivo compared to an all RNAenzymatic nucleic acid or all DNA enzyme. In some cases, the activity orstability of the nucleic acid molecule can be decreased (i.e., less thanten-fold), but the overall activity of the nucleic acid molecule isenhanced, in vivo.

[0432] By “comprising” is meant including, but not limited to, whateverfollows the word “comprising”. Thus, use of the term “comprising”indicates that the listed elements are required or mandatory, but thatother elements are optional and can or can not be present. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of”. Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements can be present.

[0433] The term “negatively charged molecules” as used herein, refers tomolecules such as nucleic acid molecules (e.g., RNA, DNA,oligonucleotides, mixed polymers, peptide nucleic acid, and the like),peptides (e.g., polyaminoacids, polypeptides, proteins and the like),nucleotides, pharmaceutical and biological compositions, that havenegatively charged groups that can ion-pair with the positively chargedhead group of the cationic lipids of the invention.

[0434] The term “coupling” as used herein, refers to a reaction, eitherchemical or enzymatic, in which one atom, moiety, group, compound ormolecule is joined to another atom, moiety, group, compound or molecule.

[0435] The terms “deprotection” or “deprotecting” as used herein, refersto the removal of a protecting group.

[0436] The term “alkyl” as used herein refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain “isoalkyl”, andcyclic alkyl groups. The term “alkyl” also comprises alkoxy, alkyl-thio,alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy,cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably it is a lower alkyl offrom about 1 to about 7 carbons, more preferably about 1 to about 4carbons. The alkyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” alsoincludes alkenyl groups containing at least one carbon-carbon doublebond, including straight-chain, branched-chain, and cyclic groups.Preferably, the alkenyl group has about 2 to about 12 carbons. Morepreferably it is a lower alkenyl of from about 2 to about 7 carbons,more preferably about 2 to about 4 carbons. The alkenyl group can besubstituted or unsubstituted. When substituted the substituted group(s)preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy,alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl,alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substitutedaryl groups. The term “alkyl” also includes alkynyl groups containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group hasabout 2 to about 12 carbons. More preferably it is a lower alkynyl offrom about 2 to about 7 carbons, more preferably about 2 to about 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moietiesof the invention can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. The preferred substituent(s)of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano,alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” grouprefers to an alkyl group (as described above) covalently joined to anaryl group (as described above). Carbocyclic aryl groups are groupswherein the ring atoms on the aromatic ring are all carbon atoms. Thecarbon atoms are optionally substituted. Heterocyclic aryl groups aregroups having from about 1 to about 3 heteroatoms as ring atoms in thearomatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

[0437] The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkylether, for example, methoxyethyl or ethoxymethyl.

[0438] The term “alkyl-thio-alkyl” as used herein refers to analkyl-S-alkyl thioether, for example, methylthiomethyl ormethylthioethyl.

[0439] The term “amino” as used herein refers to a nitrogen containinggroup as is known in the art derived from ammonia by the replacement ofone or more hydrogen radicals by organic radicals. For example, theterms “aminoacyl” and “aminoalkyl” refer to specific N-substitutedorganic radicals with acyl and alkyl substituent groups respectively.

[0440] The term “amination” as used herein refers to a process in whichan amino group or substituted amine is introduced into an organicmolecule.

[0441] The term “exocyclic amine protecting moiety” as used hereinrefers to a nucleobase amino protecting group compatible witholigonucleotide synthesis, for example, an acyl or amide group.

[0442] The term “alkenyl” as used herein refers to a straight orbranched hydrocarbon of a designed number of carbon atoms containing atleast one carbon-carbon double bond. Examples of “alkenyl” includevinyl, allyl, and 2-methyl-3-heptene.

[0443] The term “alkoxy” as used herein refers to an alkyl group ofindicated number of carbon atoms attached to the parent molecular moietythrough an oxygen bridge. Examples of alkoxy groups include, forexample, methoxy, ethoxy, propoxy and isopropoxy.

[0444] The term “alkynyl” as used herein refers to a straight orbranched hydrocarbon of a designed number of carbon atoms containing atleast one carbon-carbon triple bond. Examples of “alkynyl” includepropargyl, propyne, and 3-hexyne.

[0445] The term “aryl” as used herein refers to an aromatic hydrocarbonring system containing at least one aromatic ring. The aromatic ring canoptionally be fused or otherwise attached to other aromatic hydrocarbonrings or non-aromatic hydrocarbon rings. Examples of aryl groupsinclude, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthaleneand biphenyl. Preferred examples of aryl groups include phenyl andnaphthyl.

[0446] The term “cycloalkenyl” as used herein refers to a C3-C8 cyclichydrocarbon containing at least one carbon-carbon double bond. Examplesof cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

[0447] The term “cycloalkyl” as used herein refers to a C3-C8 cyclichydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0448] The term “cycloalkylalkyl,” as used herein, refers to a C3-C7cycloalkyl group attached to the parent molecular moiety through analkyl group, as defined above. Examples of cycloalkylalkyl groupsinclude cyclopropylmethyl and cyclopentylethyl.

[0449] The terms “halogen” or “halo” as used herein refers to indicatefluorine, chlorine, bromine, and iodine.

[0450] The term “heterocycloalkyl,” as used herein refers to anon-aromatic ring system containing at least one heteroatom selectedfrom nitrogen, oxygen, and sulfur. The heterocycloalkyl ring can beoptionally fused to or otherwise attached to other heterocycloalkylrings and/or non-aromatic hydrocarbon rings. Preferred heterocycloalkylgroups have from 3 to 7 members. Examples of heterocycloalkyl groupsinclude, for example, piperazine, morpholine, piperidine,tetrahydrofuran, pyrrolidine, and pyrazole. Preferred heterocycloalkylgroups include piperidinyl, piperazinyl, morpholinyl, and pyrolidinyl.

[0451] The term “heteroaryl” as used herein refers to an aromatic ringsystem containing at least one heteroatom selected from nitrogen,oxygen, and sulfur. The heteroaryl ring can be fused or otherwiseattached to one or more heteroaryl rings, aromatic or non-aromatichydrocarbon rings or heterocycloalkyl rings. Examples of heteroarylgroups include, for example, pyridine, furan, thiophene,5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples ofheteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl,pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl,thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl,benzisothiazolyl, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl,and benzopyrazolyl.

[0452] The term “C1-C6 hydrocarbyl” as used herein refers to straight,branched, or cyclic alkyl groups having 1-6 carbon atoms, optionallycontaining one or more carbon-carbon double or triple bonds. Examples ofhydrocarbyl groups include, for example, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene,cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl andpropargyl. When reference is made herein to C1-C6 hydrocarbyl containingone or two double or triple bonds it is understood that at least twocarbons are present in the alkyl for one double or triple bond, and atleast four carbons for two double or triple bonds.

[0453] The term “protecting group” as used herein, refers to groupsknown in the art that are readily introduced and removed from an atom,for example O, N, P, or S. Protecting groups are used to preventundesirable reactions from taking place that can compete with theformation of a specific compound or intermediate of interest. See also“Protective Groups in Organic Synthesis”, 3rd Ed., 1999, Greene, T. W.and related publications.

[0454] The term “nitrogen protecting group,” as used herein, refers togroups known in the art that are readily introduced on to and removedfrom a nitrogen. Examples of nitrogen protecting groups include Boc,Cbz, benzoyl, and benzyl. See also “Protective Groups in OrganicSynthesis”, 3rd Ed., 1999, Greene, T. W. and related publications.

[0455] The term “hydroxy protecting group,” or “hydroxy protection” asused herein, refers to groups known in the art that are readilyintroduced on to and removed from an oxygen, specifically an —OH group.Examples of hyroxy protecting groups include trityl or substitutedtrityl goups, such as monomethoxytrityl and dimethoxytrityl, orsubstituted silyl groups, such as tert-butyldimethyl, trimethylsilyl, ortert-butyldiphenyl silyl groups. See also “Protective Groups in OrganicSynthesis”, 3rd Ed., 1999, Greene, T. W. and related publications.

[0456] The term “acyl” as used herein refers to —C(O)R groups, wherein Ris an alkyl or aryl.

[0457] The term “phosphorus containing group” as used herein, refers toa chemical group containing a phosphorus atom. The phosphorus atom canbe trivalent or pentavalent, and can be substituted with O, H, N, S, Cor halogen atoms. Examples of phosphorus containing groups of theinstant invention include but are not limited to phosphorus atomssubstituted with O, H, N, S, C or halogen atoms, comprising phosphonate,alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate,phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramiditegroups, nucleotides and nucleic acid molecules.

[0458] The term “phosphine” or “phosphite” as used herein refers to atrivalent phosphorus species, for example compounds having Formula 97:

[0459] wherein R can include the groups:

[0460] and wherein S and T independently include the groups:

[0461] The term “phosphate” as used herein refers to a pentavalentphosphorus species, for example a compound having Formula 98:

[0462] wherein R includes the groups:

[0463] and wherein S and T each independently can be a sulfur or oxygenatom or a group which can include:

[0464] and wherein M comprises a sulfur or oxygen atom. The phosphate ofthe invention can comprise a nucleotide phosphate, wherein any R, S, orT in Formula 98 comprises a linkage to a nucleic acid or nucleoside.

[0465] The term “cationic salt” as used herein refers to any organic orinorganic salt having a net positive charge, for example atriethylammonium (TEA) salt.

[0466] The term “degradable linker” as used herein, refers to linkermoieties that are capable of cleavage under various conditions.Conditions suitable for cleavage can include but are not limited to pH,UV irradiation, enzymatic activity, temperature, hydrolysis,elimination, and substitution reactions, and thermodynamic properties ofthe linkage.

[0467] The term “photolabile linker” as used herein, refers to linkermoieties as are known in the art, that are selectively cleaved underparticular UV wavelengths. Compounds of the invention containingphotolabile linkers can be used to deliver compounds to a target cell ortissue of interest, and can be subsequently released in the presence ofa UV source.

[0468] The term “nucleic acid conjugates” as used herein, refers tonucleoside, nucleotide and oligonucleotide conjugates.

[0469] The term “lipid” as used herein, refers to any lipophiliccompound. Non-limiting examples of lipid compounds include fatty acidsand their derivatives, including straight chain, branched chain,saturated and unsaturated fatty acids, carotenoids, terpenes, bileacids, and steroids, including cholesterol and derivatives or analogsthereof.

[0470] The term “folate” as used herein, refers to analogs andderivatives of folic acid, for example antifolates, dihydrofloates,tetrahydrofolates, tetrahydorpterins, folinic acid, pteropolyglutamicacid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroicacid derivatives.

[0471] The term “compounds with neutral charge” as used herein, refersto compositions which are neutral or uncharged at neutral orphysiological pH. Examples of such compounds are cholesterol and othersteroids, cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidylcholine, distearoylphosphotidyl choline (DSPC), fatty acids such asoleic acid, phosphatidic acid and its derivatives, phosphatidyl serine,polyethylene glycol

[0472] —conjugated phosphatidylamine, phosphatidylcholine,phosphatidylethanolamine and related variants, prenylated compoundsincluding farnesol, polyprenols, tocopherol, and their modified forms,diacylsuccinyl glycerols, fusogenic or pore forming peptides,dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.

[0473] The term “lipid aggregate” as used herein refers to alipid-containing composition wherein the lipid is in the form of aliposome, micelle (non-lamellar phase) or other aggregates with one ormore lipids.

[0474] The term “biological system” as used herein, refers to aeukaryotic system or a prokaryotic system, can be a bacterial cell,plant cell or a mammalian cell, or can be of plant origin, mammalianorigin, yeast origin, Drosophila origin, or archebacterial origin.

[0475] The term “systemic administration” as used herein refers to thein vivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes which lead to systemic absorption include, without limitations:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexpose the desired negatively charged polymers, e.g., nucleic acids, toan accessible diseased tissue. The rate of entry of a drug into thecirculation has been shown to be a function of molecular weight or size.The use of a liposome or other drug carrier comprising the compounds ofthe instant invention can potentially localize the drug, for example, incertain tissue types, such as the tissues of the reticular endothelialsystem (RES). A liposome formulation which can facilitate theassociation of drug with the surface of cells, such as, lymphocytes andmacrophages is also useful. This approach can provide enhanced deliveryof the drug to target cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of abnormal cells, such asthe cancer cells.

[0476] The term “pharmacological composition” or “pharmaceuticalformulation” refers to a composition or formulation in a form suitablefor administration, for example, systemic administration, into a cell orpatient, preferably a human. Suitable forms, in part, depend upon theuse or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationto reach a target cell (i.e., a cell to which the negatively chargedpolymer is targeted).

[0477] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0478] The drawings will be first described briefly.

DRAWINGS

[0479]FIG. 1 shows examples of chemically stabilized ribozyme motifs. HHRz, represents hammerhead ribozyme motif (Usman et al., 1996, Curr. Op.Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig& Sproat, International PCT Publication No. WO 98/58058); G-Cleaver,represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic AcidsResearch 26, 4116-4120, Eckstein et al., International PCT publicationNo. WO 99/16871). N or n, represent independently a nucleotide which canbe same or different and have complementarity to each other; rI,represents ribo-Inosine nucleotide; arrow indicates the site of cleavagewithin the target. Position 4 of the HH Rz and the NCH Rz is shown ashaving 2′-C-allyl modification, but those skilled in the art willrecognize that this position can be modified with other modificationswell known in the art, so long as such modifications do notsignificantly inhibit the activity of the ribozyme.

[0480]FIG. 2 shows an example of the Amberzyme ribozyme motif that ischemically stabilized (see for example Beigelman et al., InternationalPCT publication No. WO 99/55857).

[0481]FIG. 3 shows an example of the Zinzyme A ribozyme motif that ischemically stabilized (see for example Beigelman et al., Beigelman etal., International PCT publication No. WO 99/55857).

[0482]FIG. 4 shows an example of a DNAzyme motif described by Santoro etal., 1997, PNAS, 94, 4262.

[0483]FIG. 5 shows a synthetic scheme for the synthesis of a folateconjugate of the instant invention.

[0484]FIG. 6 shows representative examples of fludarabine-folateconjugate molecules of the invention.

[0485]FIG. 7 shows a synthetic scheme for post-synthetic modification ofa nucleic acid molecule to produce a folate conjugate.

[0486]FIG. 8 shows a synthetic scheme for generating a protected pteroicacid synthon of the invention.

[0487]FIG. 9 shows a synthetic scheme for generating a 2-dithiopyridylactivated folic acid synthon of the invention.

[0488]FIG. 10 shows a synthetic scheme for generating an oligonucleotideor nucleic acid-folate conjugate.

[0489]FIG. 11 shows an alternative synthetic scheme for generating anoligonucleotide or nucleic acid-folate conjugate.

[0490]FIG. 12 shows an alternative synthetic scheme for post-syntheticmodification of a nucleic acid molecule to produce a folate conjugate.

[0491]FIG. 13 shows a non-limiting example of a synthetic scheme for thesynthesis of a N-acetyl-D-galactosamine-2′-aminouridine phosphoramiditeconjugate of the invention.

[0492]FIG. 14 shows a non-limiting example of a synthetic scheme for thesynthesis of a N-acetyl-D-galactosamine-D-threoninol phosphoramiditeconjugate of the invention.

[0493]FIG. 15 shows a non-limiting example of a N-acetyl-D-galactosaminesiNA nucleic acid conjugate and a N-acetyl-D-galactosamine enzymaticnucleic acid conjugate of the invention. W shown in the example refersto a biodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides. ThesiNA can be conjugated at the 3′, 5′ or both 3′ and 5′ ends of the sensestrand of a double stranded siNA and/or the 3′-end of the antisensestrand of the siNA. A single stranded siNA molecule can be conjugated atthe 3′-end of the siNA.

[0494]FIG. 16 shows a non-limiting example of a synthetic scheme for thesynthesis of a dodecanoic acid derived conjugate linker of theinvention.

[0495]FIG. 17 shows a non-limiting example of a synthetic scheme for thesynthesis of an oxime linked nucleic acid/peptide conjugate of theinvention.

[0496]FIG. 18 shows non-limiting examples of phospholipid derivednucleic acid conjugates of the invention. W shown in the examples refersto a biodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides. ThesiNA can be conjugated at the 3′, 5′ or both 3′ and 5′ ends of the sensestrand of a double stranded siNA and/or the 3′-end of the antisensestrand of the siNA. A single stranded siNA molecule can be conjugated atthe 3′-end of the siNA.

[0497]FIG. 19 shows a non-limiting example of a synthetic scheme forpreparing a phospholipid derived siNA conjugates of the invention.

[0498]FIG. 20 shows a non-limiting example of a synthetic scheme forpreparing a polyethylene glycol (PEG) derived enzymatic nucleic acidconjugates of the invention.

[0499]FIG. 21 shows PK data of a 40K PEG conjugated enzymatic nucleicacid molecule compared to the corresponding non-conjugated enzymaticnucleic acid molecule. The graph is a time course of serum concentrationin mice dosed with 30 mg/kg of Angiozyme™ or 40-kDa-PEG-Angiozyme™. Thehybridization method was used to quantitate Angiozyme™ levels.

[0500]FIG. 22 shows PK data of a phospholipid conjugated enzymaticnucleic acid molecule compared to the corresponding non-conjugatedenzymatic nucleic acid molecule.

[0501]FIG. 23 shows a non-limiting example of a synthetic scheme forpreparing a poly-N-acetyl-D-galactosamine nucleic acid conjugate of theinvention.

[0502]FIG. 24a-b shows a non-limiting example of a synthetic approachfor synthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using oxime and morpholino linkages.

[0503]FIG. 25 shows a non-limiting example of a synthetic approach forsynthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using oxime and phosphoramidate linkages.

[0504]FIG. 26a-b shows a non-limiting example of a synthetic approachfor synthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using phosphoramidate linkages.

[0505]FIG. 27 shows non-limiting examples of phospholipid derivedprotein/peptide conjugates of the invention. W shown in the examplesrefers to a biodegradable linker, for example a nucleic acid dimer,trimer, or tetramer comprising ribonucleotides and/ordeoxyribonucleotides.

[0506]FIG. 28 shows a non-limiting example of anN-acetyl-D-galactosamine peptide/protein conjugate of the invention, theexample shown is with a peptide. W shown in the example refers to abiodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides.

[0507]FIG. 29 shows a non-limiting example of a synthetic approach forsynthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using phosphoramidate linkages via coupling aprotein phosphoramidite to a PEG conjugated nucleic acid linker.

[0508]FIG. 30 shows a non-limiting example of the synthesis of siNAcholesterol conjugates of the invention using a phosphoramiditeapproach.

[0509]FIG. 31 shows a non-limiting example of the synthesis of siNA PEGconjugates of the invention using NHS ester coupling.

[0510]FIG. 32 shows a non-limiting example of the synthesis of siNAcholesterol conjugates of the invention using NHS ester coupling.

[0511]FIG. 33 shows a non-limiting example of various siNA cholesterolconjugates of the invention.

[0512]FIG. 34 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

[0513]FIG. 35 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

[0514]FIG. 36 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

[0515]FIG. 37 shows a non-limiting example of various siNA phospholipidconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

[0516]FIG. 38 shows a non-limiting example of various siNA phospholipidconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

[0517]FIG. 39 shows a non-limiting example of various siNA galactosamineconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

[0518]FIG. 40 shows a non-limiting example of various siNA galactosamineconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

[0519]FIG. 41 shows a non-limiting example of various generalized siNAconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule. CONJ in the figure refers to any biologicallyactive compound or any other conjugate compound as described herein andin the Formulae herein.

[0520]FIG. 42 shows a non-limiting example of various generalized siNAconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule. CONJ in the figure refers to any biologicallyactive compound or any other conjugate compound as described herein andin the Formulae herein.

[0521]FIG. 43 shows a non-limiting example of the pharmacokineticdistribution of intact siNA in liver after administration of conjugatedor unconjugated siNA molecules in mice.

[0522]FIG. 44 shows a non-limiting example of the activity of conjugatedsiNA constructs compared to matched chemistry unconjugated siNAconstructs in an HBV cell culture system without the use of transfectionlipid. As shown in the Figure, siNA conjugates provide efficacy in cellculture without the need for transfection reagent.

[0523]FIG. 45 shows a non-limiting example of a scheme for the synthesisof a mono-galactosamine phosphoramidite of the invention that can beused to generate galactosamine conjugated nucleic acid molecules.

[0524]FIG. 46 shows a non-limiting example of a scheme for the synthesisof a tri-galactosamine phosphoramidite of the invention that can be usedto generate tri-galactosamine conjugated nucleic acid molecules.

[0525]FIG. 47 shows a non-limiting example of a scheme for the synthesisof another tri-galactosamine phosphoramidite of the invention that canbe used to generate tri-galactosamine conjugated nucleic acid molecules.

[0526]FIG. 48 shows a non-limiting example of an alternate scheme forthe synthesis of a tri-galactosamine phosphoramidite of the inventionthat can be used to generate tri-galactosamine conjugated nucleic acidmolecules.

[0527]FIG. 49 shows a non-limiting example of a scheme for the synthesisof a cholesterol NHS ester of the invention that can be used to generatecholesterol conjugated nucleic acid molecules.

METHOD OF USE

[0528] The compositions and conjugates of the instant invention can beused to administer pharmaceutical agents. Pharmaceutical agents prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state in a patient.

[0529] Generally, the compounds of the instant invention are introducedby any standard means, with or without stabilizers, buffers, and thelike, to form a pharmaceutical composition. For use of a liposomedelivery mechanism, standard protocols for formation of liposomes can befollowed. The compositions of the present invention can also beformulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions; suspensions for injectable administration; and the like.

[0530] The present invention also includes pharmaceutically acceptableformulations of the compounds described above, preferably in combinationwith the molecule(s) to be delivered. These formulations include saltsof the above compounds, e.g., acid addition salts, for example, salts ofhydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

[0531] In one embodiment, the invention features the use of thecompounds of the invention in a composition comprising surface-modifiedliposomes containing poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes). In another embodiment,the invention features the use of compounds of the invention covalentlyattached to polyethylene glycol. These formulations offer a method forincreasing the accumulation of drugs in target tissues. This class ofdrug carriers resists opsonization and elimination by the mononuclearphagocytic system (MPS or RES), thereby enabling longer bloodcirculation times and enhanced tissue exposure for the encapsulated drug(Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem.Pharm. Bull. 1995, 43, 1005-1011). Such compositions have been shown toaccumulate selectively in tumors, presumably by extravasation andcapture in the neovascularized target tissues (Lasic et al., Science1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238,86-90). The long-circulating compositions enhance the pharmacokineticsand pharmacodynamics of therapeutic compounds, such as DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating compositions are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

[0532] The present invention also includes a composition(s) prepared forstorage or administration that includes a pharmaceutically effectiveamount of the desired compound(s) in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein. Forexample, preservatives, stabilizers, dyes and flavoring agents can beincluded in the composition. Examples of such agents include but are notlimited to sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. In addition, antioxidants and suspending agents can be included inthe composition.

[0533] A pharmaceutically effective dose is that dose required toprevent, inhibit the occurrence, or treat (alleviate a symptom to someextent, preferably all of the symptoms) of a disease state. Thepharmaceutically effective dose depends on the type of disease, thecomposition used, the route of administration, the type of mammal beingtreated, the physical characteristics of the specific mammal underconsideration, concurrent medication, and other factors which thoseskilled in the medical arts will recognize. Generally, an amount between0.1 mg/kg and 100 mg/kg body weight/day of active ingredients isadministered dependent upon potency of the negatively charged polymer.Furthermore, the compounds of the invention and formulations thereof canbe administered to a fetus via administration to the mother of a fetus.

[0534] The compounds of the invention and formulations thereof can beadministered orally, topically, parenterally, by inhalation or spray orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a nucleic acid moleculeof the invention and a pharmaceutically acceptable carrier. One or morenucleic acid molecules of the invention can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and if desired other active ingredients. Thepharmaceutical compositions containing nucleic acid molecules of theinvention can be in a form suitable for oral use, for example, astablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs.

[0535] Compositions intended for oral use can be prepared according toany method known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

[0536] Formulations for oral use can also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

[0537] Aqueous suspensions contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

[0538] Oily suspensions can be formulated by suspending the activeingredients in a vegetable oil, for example arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.The oily suspensions can contain a thickening agent, for examplebeeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoringagents can be added to provide palatable oral preparations. Thesecompositions can be preserved by the addition of an anti-oxidant such asascorbic acid.

[0539] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredient in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents or suspending agents are exemplified by those already mentionedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, can also be present.

[0540] Pharmaceutical compositions of the invention can also be in theform of oil-in-water emulsions. The oily phase can be a vegetable oil ora mineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example, sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

[0541] Syrups and elixirs can be formulated with sweetening agents, forexample glycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

[0542] The compounds of the invention can also be administered in theform of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

[0543] Compounds of the invention can be administered parenterally in asterile medium. The drug, depending on the vehicle and concentrationused, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

[0544] Dosage levels of the order of from about 0.1 mg to about 140 mgper kilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient.

[0545] It will be understood, however, that the specific dose level forany particular patient will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

[0546] For administration to non-human animals, the composition can alsobe added to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

[0547] The compounds of the present invention can also be administeredto a patient in combination with other therapeutic compounds to increasethe overall therapeutic effect. The use of multiple compounds to treatan indication can increase the beneficial effects while reducing thepresence of side effects.

[0548] Synthesis of Nucleic Acid Molecules

[0549] Synthesis of nucleic acids greater than 100 nucleotides in lengthis difficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small refers to nucleic acid motifs less than about 100 nucleotides inlength, preferably less than about 80 nucleotides in length, and morepreferably less than about 50 nucleotides in length; e.g., antisenseoligonucleotides, hammerhead or the NCH ribozymes) are preferably usedfor exogenous delivery. The simple structure of these moleculesincreases the ability of the nucleic acid to invade targeted regions ofRNA structure. Exemplary molecules of the instant invention arechemically synthesized, and others can similarly be synthesized.

[0550] Oligonucleotides (eg; antisense GeneBlocs) are synthesized usingprotocols known in the art as described in Caruthers et al., 1992,Methods in Enzymology 211, 3-19, Thompson et al., International PCTPublication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res.23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennanet al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.6,001,311. All of these references are incorporated herein by reference.The synthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. In a non-limiting example, smallscale syntheses are conducted on a 394 Applied Biosystems, Inc.synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling stepfor 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxynucleotides. Table II outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. In a non-limiting example, a 33-fold excess(60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-foldexcess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used ineach coupling cycle of 2′-O-methyl residues relative to polymer-bound5′-hydroxyl. In a non-limiting example, a 22-fold excess (40 μL of 0.11M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyltetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycleof deoxy residues relative to polymer-bound 5′-hydroxyl. Averagecoupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include but are not limited to;detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

[0551] Deprotection of the antisense oligonucleotides is performed asfollows: the polymer-bound trityl-on oligoribonucleotide is transferredto a 4 mL glass screw top vial and suspended in a solution of 40% aq.methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., thesupernatant is removed from the polymer support. The support is washedthree times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder. Standard drying or lyophilization methods known to those skilledin the art can be used.

[0552] The method of synthesis used for normal RNA including certainenzymatic nucleic acid molecules follows the procedure as described inUsman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990,Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic AcidsRes. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, andmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 7.5 min coupling step for alkylsilyl protected nucleotides and a2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlinesthe amounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesized include; detritylation solution is3% TCA in methylene chloride (ABI); capping is performed with 16%N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₁₂, 49 mMpyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson SynthesisGrade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

[0553] Deprotection of the RNA is performed using either a two-pot orone-pot protocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA·3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

[0554] Alternatively, for the one-pot protocol, the polymer-boundtrityl-on oligoribonucleotide is transferred to a 4 mL glass screw topvial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1(0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA·3HF (0.1mL) is added and the vial is heated at 65° C. for 15 min. The sample iscooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

[0555] For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 min. The cartridge is then washed again with water, salt exchangedwith 1 M NaCl and washed with water again. The oligonucleotide is theneluted with 30% acetonitrile.

[0556] Inactive hammerhead ribozymes or binding attenuated control((BAC) oligonucleotides) are synthesized by substituting a U for G₅ anda U for A₁₄ (numbering from Hertel, K. J., et al., 1992, Nucleic AcidsRes., 20, 3252). Similarly, one or more nucleotide substitutions can beintroduced in other enzymatic nucleic acid molecules to inactivate themolecule and such molecules can serve as a negative control.

[0557] The average stepwise coupling yields are typically ≧98% (Wincottet al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skillin the art will recognize that the scale of synthesis can be adapted tobe larger or smaller than the example described above including, but notlimited to, 96 well format, with the ratio of chemicals used in thereaction being adjusted accordingly.

[0558] Alternatively, the nucleic acid molecules of the presentinvention can be synthesized separately and joined togetherpost-synthetically, for example by ligation (Moore et al., 1992, Science256, 9923; Draper et al., International PCT publication No. WO 93/23569;Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al.,1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997,Bioconjugate Chem. 8, 204).

[0559] The nucleic acid molecules of the present invention are modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). Nucleic acid conjugaes of theinvention can purified by gel electrophoresis using general methods orare purified by high pressure liquid chromatography (HPLC; See Wincottet al., Supra, the totality of which is hereby incorporated herein byreference) or hydrophobic interaction chromatography and arere-suspended in water.

[0560] Optimizing Activity of the Nucleic Acid Molecule of theInvention.

[0561] Chemically synthesizing nucleic acid molecules with modifications(base, sugar and/or phosphate) that prevent their degradation by serumribonucleases can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al.,supra; all of these describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules herein). Modifications which enhance their efficacy in cells,and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired. (All these publications are hereby incorporated by referenceherein).

[0562] There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro,2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; allof the references are hereby incorporated in their totality by referenceherein). Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into ribozymes without inhibiting catalysis,and are incorporated by reference herein. In view of such teachings,similar modifications can be used as described herein to modify thenucleic acid molecules of the instant invention.

[0563] While chemical modification of oligonucleotide internucleotidelinkages with phosphorothioate, phosphorothioate, and/or5′-methylphosphonate linkages improves stability, too many of thesemodifications may cause some toxicity. Therefore, when designing nucleicacid molecules the amount of these internucleotide linkages should beminimized. Without being bound by any particular theory, the reductionin the concentration of these linkages should lower toxicity resultingin increased efficacy and higher specificity of these molecules.

[0564] Nucleic acid molecules having chemical modifications thatmaintain or enhance activity are provided. Such nucleic acid is alsogenerally more resistant to nucleases than unmodified nucleic acid.Thus, in a cell and/or in vivo the activity can not be significantlylowered. Therapeutic nucleic acid molecules (e.g., enzymatic nucleicacid molecules and antisense nucleic acid molecules) deliveredexogenously are optimally stable within cells until translation of thetarget RNA has been inhibited long enough to reduce the levels of theundesirable protein. This period of time varies between hours to daysdepending upon the disease state. The nucleic acid molecules should beresistant to nucleases in order to function as effective intracellulartherapeutic agents. Improvements in the chemical synthesis of RNA andDNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al.,1992, Methods in Enzymology 211,3-19 (incorporated by reference herein)have expanded the ability to modify nucleic acid molecules byintroducing nucleotide modifications to enhance their nuclease stabilityas described above.

[0565] Use of the nucleic acid-based molecules of the invention can leadto better treatment of the disease progression by affording thepossibility of combination therapies (e.g., multiple antisense orenzymatic nucleic acid molecules targeted to different genes, nucleicacid molecules coupled with known small molecule inhibitors, orintermittent treatment with combinations of molecules (includingdifferent motifs) and/or other chemical or biological molecules). Thetreatment of patients with nucleic acid molecules can also includecombinations of different types of nucleic acid molecules.

[0566] In another embodiment, nucleic acid catalysts having chemicalmodifications that maintain or enhance enzymatic activity are provided.Such nucleic acids are also generally more resistant to nucleases thanunmodified nucleic acid. Thus, in a cell and/or in vivo the activity ofthe nucleic acid can not be significantly lowered. As exemplified hereinsuch enzymatic nucleic acids are useful in a cell and/or in vivo even ifactivity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry,35, 14090). Such enzymatic nucleic acids herein are said to “maintain”the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.

[0567] In another aspect the nucleic acid molecules comprise a 5′ and/ora 3′-cap structure.

[0568] In another embodiment the 3′-cap includes, for example4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridgingor non bridging methylphosphonate and 5′-mercapto moieties (for moredetails see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporatedby reference herein).

[0569] In one embodiment, the invention features modified enzymaticnucleic acid molecules with phosphate backbone modifications comprisingone or more phosphorothioate, phosphorodithioate, methylphosphonate,morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide,sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/oralkylsilyl, substitutions. For a review of oligonucleotide backbonemodifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues:Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, andMesmaeker et al., 1994, Novel Backbone Replacements forOligonucleotides, in Carbohydrate Modifications in Antisense Research,ACS, 24-39. These references are hereby incorporated by referenceherein.

[0570] In connection with 2′-modified nucleotides as described for theinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO98/28317, respectively, which are both incorporated by reference intheir entireties.

[0571] Various modifications to nucleic acid (e.g., antisense andribozyme) structure can be made to enhance the utility of thesemolecules. For example, such modifications can enhance shelf-life,half-life in vitro, stability, and ease of introduction of sucholigonucleotides to the target site, including e.g., enhancingpenetration of cellular membranes and conferring the ability torecognize and bind to targeted cells.

[0572] Use of these molecules can lead to better treatment of diseaseprogression by affording the possibility of combination therapies (e.g.,multiple enzymatic nucleic acid molecules targeted to different genes,enzymatic nucleic acid molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations of enzymaticnucleic acid molecules (including different enzymatic nucleic acidmolecule motifs) and/or other chemical or biological molecules). Thetreatment of patients with nucleic acid molecules can also includecombinations of different types of nucleic acid molecules. Therapies canbe devised which include a mixture of enzymatic nucleic acid molecules(including different enzymatic nucleic acid molecule motifs), antisenseand/or 2-5A chimera molecules to one or more targets to alleviatesymptoms of a disease.

[0573] Indications

[0574] Particular disease states that can be treated using compounds andcompositions of the invention include, but are not limited to, cancersand cancerous conditions such as breast, lung, prostate, colorectal,brain, esophageal, stomach, bladder, pancreatic, cervical,hepatocellular, head and neck, and ovarian cancer, melanoma, lymphoma,glioma, multidrug resistant cancers; ocular conditions such as maculardegeneration and diabetic retinopathy, and/or viral infections includingHIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus, westnile virus, severe acute respiratory syndrome (SARS) virus, Ebola virus,foot and mouth virus, and papilloma virus infection.

[0575] The molecules of the invention can be used in conjunction withother known methods, therapies, or drugs. For example, the use ofmonoclonal antibodies (eg; mAb IMC C225, mAB ABX-EGF) treatment,tyrosine kinase inhibitors (TKIs), for example OSI-774 and ZD1839,chemotherapy, and/or radiation therapy, are all non-limiting examples ofa methods that can be combined with or used in conjunction with thecompounds of the instant invention. Common chemotherapies that can becombined with nucleic acid molecules of the instant invention includevarious combinations of cytotoxic drugs to kill the cancer cells. Thesedrugs include, but are not limited to, paclitaxel (Taxol), docetaxel,cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracilcarboplatin, edatrexate, gemcitabine, vinorelbine etc. Those skilled inthe art will recognize that other drug compounds and therapies can besimilarly be readily combined with the compounds of the instantinvention are hence within the scope of the instant invention.

[0576] Diagnostic Uses

[0577] The compounds of this invention, for example, nucleic acidconjugate molecules, can be used as diagnostic tools to examine geneticdrift and mutations within diseased cells or to detect the presence of adisease related RNA in a cell. The close relationship between, forexample, enzymatic nucleic acid molecule activity and the structure ofthe target RNA allows the detection of mutations in any region of themolecule which alters the base-pairing and three-dimensional structureof the target RNA. By using multiple enzymatic nucleic acid moleculesconjugates of the invention, one can map nucleotide changes which areimportant to RNA structure and function in vitro, as well as in cellsand tissues. Cleavage of target RNAs with enzymatic nucleic acidmolecules can be used to inhibit gene expression and define the role(essentially) of specified gene products in the progression of disease.In this manner, other genetic targets can be defined as importantmediators of the disease. These experiments can lead to better treatmentof the disease progression by affording the possibility of combinationaltherapies (e.g., multiple enzymatic nucleic acid molecules targeted todifferent genes, enzymatic nucleic acid molecules coupled with knownsmall molecule inhibitors, or intermittent treatment with combinationsof enzymatic nucleic acid molecules and/or other chemical or biologicalmolecules). Other in vitro uses of enzymatic nucleic acid molecules ofthis invention are well known in the art, and include detection of thepresence of mRNAs associated with a disease-related condition. Such RNAis detected by determining the presence of a cleavage product aftertreatment with an enzymatic nucleic acid molecule using standardmethodology.

[0578] In a specific example, enzymatic nucleic acid molecules that aredelivered to cells as conjugates and which cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstenzymatic nucleic acid molecule is used to identify wild-type RNApresent in the sample and the second enzymatic nucleic acid molecule isused to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth enzymatic nucleic acid molecules to demonstrate the relativeenzymatic nucleic acid molecule efficiencies in the reactions and theabsence of cleavage of the “non-targeted” RNA species. The cleavageproducts from the synthetic substrates also serve to generate sizemarkers for the analysis of wild-type and mutant RNAs in the samplepopulation. Thus each analysis requires two enzymatic nucleic acidmolecules, two substrates and one unknown sample which is combined intosix reactions. The presence of cleavage products is determined using anRNAse protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype is adequate to establishrisk. If probes of comparable specific activity are used for bothtranscripts, then a qualitative comparison of RNA levels will beadequate and will decrease the cost of the initial diagnosis. Highermutant form to wild-type ratios are correlated with higher risk whetherRNA levels are compared qualitatively or quantitatively. The use ofenzymatic nucleic acid molecules in diagnostic applications contemplatedby the instant invention is more fully described in George et al., U.S.Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332,Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,International PCT publication No. WO 00/24931, Breaker et al.,International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., International PCT publication No. WO 99/29842.

[0579] Additional Uses

[0580] Potential uses of sequence-specific enzymatic nucleic acidmolecules of the instant invention that are delivered to cells asconjugates can have many of the same applications for the study of RNAthat DNA restriction endonucleases have for the study of DNA (Nathans etal., 1975 Ann. Rev. Biochem. 44:273). For example, the pattern ofrestriction fragments can be used to establish sequence relationshipsbetween two related RNAs, and large RNAs can be specifically cleaved tofragments of a size more useful for study. The ability to engineersequence specificity of the enzymatic nucleic acid molecule is ideal forcleavage of RNAs of unknown sequence. Applicant has described the use ofnucleic acid molecules to down-regulate gene expression of target genesin bacterial, microbial, fungal, viral, and eukaryotic systems includingplant, or mammalian cells.

EXAMPLE 1 Synthesis ofO′-(4-monomethoxytrityl)-N-(6-(N-(α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate 3′-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20)(FIG. 5)

[0581] General. All reactions were carried out under a positive pressureof argon in anhydrous solvents. Commercially available reagents andanhydrous solvents were used without further purification. ¹H (400.035MHz) and ³¹P (161.947 MHz) NMR spectra were recorded in CDCl₃, unlessstated otherwise, and chemical shifts in ppm refer to TMS and H₃PO₄,respectively. Analytical thin-layer chromatography (TLC) was performedwith Merck Art.5554 Kieselgel 60 F₂₅₄ plates and flash columnchromatography using Merck 0.040-0.063 mm silica gel 60.

[0582] N-(N-Fmoc-6-aminocaproyl)-D-threoninol (13).N-Fmoc-6-aminocaproic acid (10 g, 28.30 mmol) was dissolved in DMF (50ml) and N-hydroxysuccinimide (3.26 g, 28.30 mmol) and1,3-dicyclohexylcarbodiimide (5.84 g, 28.3 mmol) were added to thesolution. The reaction mixture was stirred at RT (about 23° C.)overnight and the precipitated 1,3-dicyclohexylurea filtered off. To thefiltrate D-threoninol (2.98 g, 28.30 mmol) was added and the reactionmixture stirred at RT overnight. The solution was reduced to ca half thevolume in vacuo, the residue diluted with about m ml of ethyl acetateand extracted with about x ml of 5% NaHCO₃, followed by washing withbrine. The organic layer was dried (Na₂SO₄), evaporated to a syrup andchromatographed by silica gel column chromatography using 1-10% gradientof methanol in ethyl acetate. Fractions containing the product werepooled and evaporated to a white solid (9.94 g, 80%). ¹H-NMR(DMSO-d₆-D₂O) δ7.97-7.30 (m, 8H, aromatic), 4.34 (d, J=6.80, 2H, Fm),4.26 (t, J=6.80, 1H, Fm), 3.9 (m, 1H, H3 Thr), 3.69 (m, 1H, H2 Thr),3.49 (dd, J=10.6, J=7.0, 1H, H1 Thr), 3.35 (dd, J=10.6, J=6.2, 1H, H1′Thr), 3.01 (m, 2H, CH₂CO Acp), 2.17 (m, 2H, CH₂NH Acp), 1.54 (m, 2H, CH₂Acp), 1.45 (m, 2H, CH₂ Acp), 1.27 (m, 2H, CH₂ Acp), 1.04 (d, J=6.4, 3H,CH₃). MS/ESI⁺ m/z 441.0 (M+H)⁺.

[0583] O′-(4-Monomethoxytrityl)-N-(N-Fmoc-6-aminocaproyl)-D-threoninol(14). To the solution of 13 (6 g, 13.62 mmol) in dry pyridine (80 ml)p-anisylchlorodiphenyl-methane (6 g, 19.43 mmol) was added and thereaction mixture stirred at RT overnight. Methanol was added (20 ml) andthe solution concentrated in vacuo. The residual syrup was partitionedbetween about x ml of dichloromethane and about x ml of 5% NaHCO₃, theorganic layer was washed with brine, dried (Na₂SO₄) and evaporated todryness. Flash column chromatography using 1-3% gradient of methanol indichloromethane; afforded 14 as a white foam (6 g, 62%). ¹H-NMR (DMSO)δ7.97-6.94 (m, 22H, aromatic), 4.58 (d, 1H, J=5.2, OH), 4.35 (d, J=6.8,2H, Fm), 4.27 (t, J=6.8, 1H, Fm), 3.97 (m, 2H, H2, H3 Thr), 3.80 (s, 3H,OCH₃), 3.13 (dd, J=8.4, J=5.6, 1H, H1 Thr), 3.01 (m, 2H, CH₂CO Acp),2.92 (m, dd, J=8.4, J=6.4, 1H, H1′ Thr), 2.21 (m, 2H, CH₂NH Acp), 1.57(m, 2H, CH₂ Acp), 1.46 (m, 2H, CH₂ Acp), 1.30 (m, 2H, CH₂ Acp), 1.02 (d,J=5.6, 3H, CH₃). MS/ESI⁺ m/z 735.5 (M+Na)⁺.

[0584] O¹-(4-Monomethoxytrityl)-N-(6-aminocaproyl)-D-threoninol (15). 14(9.1 g, 12.77 mmol) was dissolved in DMF (100 ml) containing piperidine(10 ml) and the reaction mixture was kept at RT for about 1 hour. Thesolvents were removed in vacuo and the residue purified by silica gelcolumn chromatography using 1-10% gradient of methanol indichloromethane to afford 15 as a syrup (4.46 g, 71%). ¹H-NMR δ7.48-6.92(m, 14H, aromatic), 6.16 (d, J=8.8, 1H, NH), 4.17 (m, 1H, H3 Thr), 4.02(m, 1H, H2 Thr), 3.86 (s, 3H, OCH₃), 3.50 (dd, J=9.7, J=4.4, 1H, H1Thr), 3.37 (dd, J=9.7, J=3.4, 1H, H1′ Thr), 2.78 (t, J=6.8, 2H, CH₂COAcp), 2.33 (t, J=7.6, 2H, CH₂NH Acp), 1.76 (m, 2H, CH₂ Acp), 1.56 (m,2H, CH₂ Acp), 1.50 (m, 2H, CH₂ Acp), 1.21 (d, J=6.4, 3H, CH₃).MS/ESI⁺m/z 491.5 (M+H)⁺.

[0585] O′-(4-Monomethoxytrityl)-N-(6-(N-(N-Boc-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol (16). To the solution ofN-Boc-α-OFm-glutamic acid (Bachem) (1.91 g, 4.48 mmol) in DMF (10 ml)N-hydroxysuccinimide (518 mg, 4.50 mmol) and1,3-dicyclohexylcarbodiimide (928 mg, 4.50 mmol) was added and thereaction mixture was stirred at RT overnight. 1,3-Dicyclohexylurea wasfiltered off and to the filtrate 15 (2 g, 4.08 mmol) and pyridine (2 ml)were added. The reaction mixture was stirred at RT for 3 hours and thanconcentrated in vacuo. The residue was partitioned between ethyl acetateand 5% Na₂HCO₃, the organic layer extracted with brine as previouslydescribed, dried (Na₂SO₄) and evaporated to a syrup. Columnchromatography using 2-10% gradient of methanol in dichlotomethaneafforded 16 as a white foam (3.4 g, 93%). ¹H-NMR δ 7.86-6.91 (m, 22H,aromatic), 6.13 (d, J=8.8, 1H, NH), 5.93 (br s, 1H, NH), 5.43 (d, J=8.4,1H, NH), 4.63 (dd, J=10.6, J=6.4, 1H, Fm), 4.54 (dd, J=10.6, J=6.4, 1H,Fm), 4.38 (m, 1H, Glu), 4.3 (t, J=6.4, 1H, Fm), 4.18 (m, 1H, H3 Thr),4.01 (m, 1H, H2 Thr), 3.88 (s, 3H, OCH₃), 3.49 (dd, J=9.5, J=4.4, 1H, H1Thr), 3.37 (dd, J=9.5, J=3.8, 1H, H1′ Thr), 3.32 (m, 2H, CH₂CO Acp),3.09 (br s, 1H, OH), 2.32 (m, 2H, CH₂NH Acp), 2.17 (m, 3H, Glu), 1.97(m, 1H, Glu), 1.77 (m, 2H, CH₂ Acp), 1.61 (m, 2H, CH₂ Acp), 1.52 (s, 9H,t-Bu), 1.21 (d, J=6.4, 3H, CH₃). MS/ESI⁺ m/z 920.5 (M+Na)⁺.

[0586] N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninolhydrochloride (17). 16 (2 g, 2.23 mmol) was dissolved in methanol (30ml) containing anisole (10 ml) and to this solution x ml of 4M HCl indioxane was added. The reaction mixture was stirred for 3 hours at RTand then concentrated in vacuo. The residue was dissolved in ethanol andthe product precipitated by addition of x ml of ether. The precipitatewas washed with ether and dried to give 17 as a colorless foam (1 g,80%). ¹H-NMR (DMSO-d₆-D₂O) 67.97-7.40 (m, 8H, aromatic), 4.70 (m, 1H,Fm), 4.55 (m, 1H, Fm), 4.40 (t, J=6.4, 1H, Fm), 4.14 (t, J=6.6, 1H,Glu), 3.90 (dd, J=2.8, J=6.4, 1H, H3 Thr), 3.68 (m, 1H, H2 Thr), 3.49(dd, J=10.6, J=7.0, 1H, H1 Thr), 3.36 (dd, J=10.6, J=6.2, 1H, H1′ Thr),3.07 (m, 2H, CH₂CO Acp), 2.17 m, 3H), 1.93 (m, 2H), 1.45 (m, 2H), 1.27(m, 2H), 1.04 (d, J=6.4, 3H Thr). MS/ESI⁺ m/z 526.5 (M+H)⁺.

[0587]N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate (18). To the solution of N²-iBu-N¹⁰-TFA-pteroic acid¹(480 mg, 1 mmol) in DMF (5 ml) 1-hydroxybenzotriazole (203 mg, 1.50mmol), EDCI (288 mg, 1.50 mmol) and 17 (free base, 631 mg, 1.2 mmol) areadded. The reaction mixture is stirred at RT for 2 hours, thenconcentrated to ca 3 ml and loaded on the column of silica gel. Elutionwith dichloromethane, followed by 1-20% gradient of methanol indichloromethane afforded 18 (0.5 g, 51%). ¹H-NMR (DMSO-d₆-D₂O) δ 9.09(d, J=6.8, 1H, NH) 8.96 (s, 1H, H7 pteroic acid), 8.02-7.19 (m, 13H,aromatic, NH), 5.30 (s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.41 (d,J=6.8, 2H, Fm), 4.29 (t, J=6.8, 1H, Fm), 3.89 (dd, J=6.2, J=2.8, 1H, H3Thr), 3.68 (m, 1H, H2 Thr), 3.48 (dd, J=10.4, J=7.0, 1H, H1 Thr), 3.36(dd, J=10.4, J=6.2, 1H H1′ Thr), 3.06 (m, 2H, CH₂CO Acp), 2.84 (m, 1H,iBu), 2.25 (m, 2H, CH₂NH Acp), 2.16 (m, 3H, Glu), 1.99 (m, 1H, Glu),1.52 (m, 2H Acp), 1.42 (m, 2H Acp), 1.27 (m, 2H Acp), 1.20 (s, 3H iBu),1.19 (s, 3H, iBu), 1.03 (d, J=6.2, 3H Thr). MS/ESI⁻ m/z 984.5 (M−H)⁻.

[0588]O¹-(4-monomethoxytrityl)-N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate (19). To the solution of conjugate 18 (1 g, 1.01 mmol) indry pyridine (15 ml) p-anisylchlorodiphenylmethane (405 mg) was addedand the reaction mixture was stirred, protected from moisture, at RTovernight. Methanol (3 ml) was added and the reaction mixtureconcentrated to a syrup in vacuo. The residue was partitioned betweendichloromethane and 5% NaHCO₃, the organic layer washed with brine,dried (Na₂SO₄) and evaporated to dryness. Column chromatography using0.5-10% gradient of methanol in dichloromethane afforded 19 as acolorless foam (0.5 g, 39%. ¹H-NMR (DMSO-d₆-D₂O δ9.09 (d, J=6.8, 1H, NH)8.94 (s, 1H, H7 pteroic acid), 8.00-6.93 (m, 27H, aromatic, NH), 5.30(s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.40 (d, J=6.8, 2H, Fm), 4.29(t, J=6.8, 1H, Fm), 3.94 (m, 2H, H3, H2 Thr), 3.79 (s, 3H, OCH₃) 3.11(dd, J=8.6, J=5.8, 1H, H1 Thr), 3.04 (m, 2H, CH₂CO Acp), 2.91 (dd,J=8.6, J=6.4, 1H, H1′ Thr), 2.85 (m, 1H, iBu), 2.25 (m, 2H, CH₂NH Acp),2.19 (m, 2H, Glu), 2.13 (m, 1H, Glu), 1.98 (m, 1H, Glu), 1.55 (m, 2HAcp), 1.42 (m, 2H Acp), 1.29(m, 2H Acp), 1.20 (s, 3H iBu), 1.18 (s, 3H,iBu), 1.00 (d, J=6.4, 3H Thr). MS/ESI⁻ m/z 1257.0 (M−H)⁻.

[0589]O¹-(4-monomethoxytrityl)-N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate 3′-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20).To the solution of 19 (500 mg, 0.40 mmol) in dichloromethane (2 ml)2-cyanoethyl tetraisopropylphosphordiamidite (152 μL, 0.48 mmol) wasadded followed by pyridinium trifluoroacetate (93 mg, 0.48 mmol). Thereaction mixture was stirred at RT for 1 hour and than loaded on thecolumn of silica gel in hexanes. Elution using ethyl acetate-hexanes1:1, followed by ethyl acetate and ethyl acetate-acetone 1:1 in thepresence of 1% pyridine afforded 20 as a colorless foam (480 mg, 83%).³¹p NMR δ 149.4 (s), 149.0 (s).

EXAMPLE 2 Synthesis of 2-dithiopyridyl Activated Folic Acid (30) (FIG.9)

[0590] Synthesis of the cysteamine modified folate 30 is presented inFIG. 9. Monomethoxytrityl cysteamine 21 was prepared by selectivetritylation of the thiol group of cysteamine with 4-methoxytritylalcohol in trifluoroacetic acid. Peptide coupling of 21 withFmoc-Glu-OtBu (Bachem Bioscience Inc., King of Prussia, Pa.) in thepresence of PyBOP yielded 22 in a high yield. N-Fmoc group was removedsmoothly with piperidine to give 23. Condensation of 23 withp-(4-methoxytrityl)aminobenzoic acid, prepared by reaction ofp-aminobenzoic acid with 4-methoxytrityl chloride in pyridine, affordedthe fully protected conjugate 24. Selective cleavage of N-MMTr groupwith acetic acid afforded 25 in quantitative yield. Shiff base formationbetween 25 and N²-iBu-6-formylpterin 26,⁹ followed by reduction withborane-pyridine complex proceeded with a good yield to give fullyprotected cysteamine-folate adduct 27.¹² The consecutive cleavage ofprotecting groups of 27 with base and acid yielded thiol derivative 29.The thiol exchange reaction of 29 with 2,2-dipyridyl disulfide affordedthe desired S-pyridyl activated synthon 30 as a yellow powder; Isolatedas a TEA⁺ salt: ¹H NMR spectrum for 10 in D₂O: δ 8.68 (s, 1H, H-7), 8.10(d, J=3.6, 1H, pyr), 7.61 (d, J=8.8, 2H, PABA), 7.43 (m, 1H, pyr), 7.04(d, J=7.6, 1H, pyr), 6.93 (m, 1H, pyr), 6.82 (d, J=8.8, 1H, PABA), 4.60(s, 2H, 6-CH₂), 4.28 (m, 1H, Glu), 3.30-3.08 (m, 2H, cysteamine), 3.05(m, 6H, TEA), 2.37 (m, 2H, cysteamine), 2.10 (m, 4H, Glu), 1.20 (m, 9H,TEA). MS/ESI⁻ m/z 608.02 [M−H]⁻. It is worth noting that the isolationof 30 as its TEA⁺ or Na⁺ salt made it soluble in DMSO and/or water,which is an important requirement for its use in conjugation reactions.

EXAMPLE 3 Post Synthetic Conjugation of Enzymatic Nucleic Acid to FormNucleic Acid-Folate Conjugate (33) (FIG. 10)

[0591] Oligonucleotide synthesis, deprotection and purification wasperformed as described herein. 5′-Thiol-Modifier C6 (Glen Research,Sterling, Va.) was coupled as the last phosphoramidite to the 5′-end ofa growing oligonucleotide chain. After cleavage from the solid supportand base deprotection, the disulfide modified enzymatic nucleic acidmolecule 31 (FIG. 10) was purified using ion exchange chromatography.The thiol group was unmasked by reduction with dithiothreitol (DTT) toafford 32 which was purified by gel filtration and immediatelyconjugated with 30. The resulting conjugate 33 was separated from theexcess folate by gel filtration and then purified by RP HPLC usinggradient of acetonitrile in 50 mM triethylammonium acetate (TEAA).Desalting was performed by RP HPLC. Reactions were conducted on 400 mgof disulfide modified enzymatic nucleic acid molecule 31 to afford200-250 mg (50-60% yield) of conjugate 33. MALDI TOF MS confirmed thestructure: 13 [M−H]-12084.74 (calc.12083.82). An alternative approach tothis synthesis is shown in FIG. 11.

[0592] As shown in Examples 2 and 3, a folate-cysteamine adduct can beprepared by a scaleable solution phase synthesis in a good overallyield. Disulfide conjugation of this novel targeting ligand to thethiol-modified oligonucleotide is suitable for the multi-gram scalesynthesis. The 9-atom spacer provides a useful spatial separationbetween folate and attached oligonucleotide cargo. Importantly,conjugation of folate to the oligonucleotide through a disulfide bondshould permit intermolecular separation which was suggested to berequired for the functional cytosolic entry of a protein drug.

EXAMPLE 4 Synthesis of Galactose and N-acetyl-Galactosamine Conjugates(FIGS. 13, 14, and 15)

[0593] Applicant has designed both nucleoside andnon-nucleoside-N-acetyl-D-galactosamine conjugates suitable forincorporation at any desired position of an oligonucleotide. Multipleincorporations of these monomers could result in a “glycoside clustereffect”.

[0594] All reactions were carried out under a positive pressure of argonin anhydrous solvents. Commercially available reagents and anhydroussolvents were used without further purification.N-acetyl-D-galactosamine was purchased from Pfanstiel (Waukegan, Ill.),folic acid from Sigma (St. Louis, Mo.), D-threoninol from Aldrich(Milwaukee, Wis.) and N-Boc-α-OFm glutamic acid from Bachem. ¹H (400.035MHz) and 31p (161.947 MHz) NMR spectra were recorded in CDCl₃, unlessstated otherwise, and chemical shifts in ppm refer to TMS and H3PO4,respectively. Analytical thin-layer chromatography (TLC) was performedwith Merck Art.5554 Kieselgel 60 F₂₅₄ plates and flash columnchromatography using Merck 0.040-0.063 mm silica gel 60. The generalprocedures for RNA synthesis, deprotection and purification aredescribed herein. MALDI-TOF mass spectra were determined on PerSeptiveBiosystems Voyager spectrometer. Electrospray mass spectrometry was runon the PE/Sciex AP1365 instrument.

[0595] 2′-(N-L-lysyl)amino-5′-O-4,4′-dimethoxytrityl-2′-deoxyuridine(2).2′-(N-α,ε-bis-Fmoc-L-lysyl)amino-5′-O-4,4′-dimethoxytrityl-2′-deoxyuridine(1) (4 g, 3.58 mmol) was dissolved in anhydrous DMF (30 ml) anddiethylamine (4 ml) was added. The reaction mixture was stirred at rtfor 5 hours and than concentrated (oil pump) to a syrup. The residue wasdissolved in ethanol and ether was added to precipitate the product (1.8g, 75%). ¹H-NMR (DMSO-d₆-D2O) ^(δ)7.70 (d, ^(J)6,5=8.4, 1H, H6),7.48-6.95 (m, 13H, aromatic), 5.93 (d, J1′, 2′=8.4, 1H, H1′), 5.41 (d,^(J) _(5,6)=8.4, 1H, H5), 4,62 (m, 1H, H2′), 4.19 (d, 1H, ^(J)_(3′,2′)=6.0, H3′), 3.81 (s, 6H, 2×OMe), 3.30 (m, 4H, 2H5′, CH₂),1.60-1.20 (m, 6H, 3×CH₂). MS/ESI⁺ m/z 674.0 (M+H)⁺.

[0596] N-Acetyl-1,4,6-tri-O-acetyl-2-amino-2-deoxy-β-D-galactospyranose(3). N-Acetyl-D-galac-tosamine (6.77 g, 30.60 mmol) was suspended inacetonitrile (200 ml) and triethylamine (50 ml, 359 mmol) was added. Themixture was cooled in an ice-bath and acetic anhydride (50 ml, 530mmol)) was added dropwise under cooling. The suspension slowly clearedand was then stirred at rt for 2 hours. It was than cooled in anice-bath and methanol (60 ml) was added and the stirring continued for15 min. The mixture was concentrated under reduced pressure and theresidue partitioned between dichloromethane and 1 N HCl. Organic layerwas washed twice with 5% NaHCO₃, followed by brine, dried (Na2SO4) andevaporated to dryness to afford 10 g (84%) of 3 as a colorless foam. ¹HNMR was in agreement with published data (Findeis, 1994, Int. J. PeptideProtein Res., 43, 477-485.

[0597] 2-Acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactospyranose (4).This compound was prepared from 3 as described by Findeis supra.

[0598] Benzyl 12-Hydroxydodecanoate (5). To a cooled (0° C.) and stirredsolution of 12-hydroxydodecanoic acid (10.65 g, 49.2 mmol) in DMF (70ml) DBU (8.2 ml, 54.1 mmol) was added, followed by benzyl bromide (6.44ml, 54.1 mmol). The mixture was left overnight at rt, than concentratedunder reduced pressure and partitioned between 1 N HCl and ether.Organic phase was washed with saturated NaHCO₃, dried over Na₂SO₄ andevaporated. Flash chromatography using 20-30% gradient of ethyl acetatein hexanes afforded benzyl ester as a white powder (14.1 g, 93.4%).¹H-NMR spectral data were in accordance with the published values.³³

[0599] 12′-Benzylhydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyrano-se(6). 1-Chloro sugar 4 (4.26 g, 11.67 mmol) and benzyl12-hydroxydodecanoate (5) (4.3 g, 13.03 mmol) were dissolved innitromethane-toluene 1:1 (122 ml) under argon and Hg(CN)₂ (3.51 g, 13.89mmol) and powdered molecular sieves 4A (1.26 g) were added. The mixturewas stirred at rt for 24 h, filtered and the filtrate concentrated underreduced pressure. The residue was partitioned between dichloromethaneand brine, organic layer was washed with brine, followed by 0.5 M KBr,dried (Na₂SO₄) and evaporated to a syrup. Flash silica gel columnchromatography using 15-30% gradient of acetone in hexanes yieldedproduct 6 as a colorless foam (6 g, 81%). ¹H-NMR ^(δ) 7.43 (m, ^(5H),phenyl), 5.60 (d, 1H, J_(NH,2)=8.8, NH), 5.44 (d, J_(4,3)=3.2, 1H, H4),5.40 (dd, J_(3,4)=3.2, J_(3,2)=10.8, 1H, H3), 5.19 (s, 2H, CH₂Ph), 4.80(d, J_(1,2)=8.0, 1H, H1), 4.23 (m, 2H, CH₂), 3.99 (m, 3H, H2, H6), 3.56(m, 1H, H5), 2.43 (t, J=7.2, 2H, CH₂), 2.22 (s, 3H, Ac), 2.12 (s, 3H,Ac), 2.08 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.64 (m, 4H, 2×CH₂), 1.33 (brm, 14H, 7×CH₂). MS/ESI⁻ m/z 634.5 (M−H)⁻.

[0600]12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose(7).

[0601] Conjugate 6 (2 g, 3.14 mmol)) was dissolved in ethanol (50 ml)and 5% Pd-C (0.3 g) was added. The reaction mixture was hydrogenatedovernight at 45 psi H₂, the catalyst was filtered off and the filtrateevaporated to dryness to afford pure 7 (1.7 g, quantitative) as a whitefoam. ¹H-NMR ^(δ) 5.73 (d, 1H, J_(NH,2)=8.4, NH), 5.44 (d, J_(4,3)=3.0,1H, H4), 5.40 (dd, ^(J) _(3,4)=3.0, J_(3.2)=11.2,1H, H3), 4.78 (d,J_(1,2)=8.8, 1H, H1), 4.21 (m, 2H, CH₂), 4.02 (m, 3H, H2, H6), 3.55 (m,1H, H5), 2.42 (m, 2H, CH₂), 2.23 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.09 (s,3H, Ac), 2.04 (s, 3H, Ac), 1.69 (m, 4H, 2×CH₂), 1.36 (br m, 14H, 7×CH₂).MS/ESI⁻ m/z 544.0 (M−H)⁻.

[0602] 2′-(N-α,ε-bis-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose)-L-lysyl)amino-2′-deoxy-5′-O-4,4′-dimethoxytrityluridine (9). 7 (1.05 g, 1.92 mmol) was dissolved in anhydrous THF andN-hydroxysuccinimide (0.27 g, 2.35 mmol) and1,3-dicyclohexylcarbodiimide (0.55 g, 2.67 mmol) were added. Thereaction mixture was stirred at rt overnight, then filtered throughCelite pad and the filtrate concentrated under reduced pressure. Thecrude NHSu ester 8 was dissolved in dry DMF (13 ml) containingdiisopropylethylamine (0.67 ml, 3.85 mmol) and to this solutionnucleoside 2 (0.64 g, 0.95 mmol was added). The reaction mixture wasstirred at rt overnight and than concentrated under reduced pressure.The residue was partitioned between water and dichloromethane, theaqueous layer extracted with dichloromethane, the organic layerscombined, dried (Na₂SO₄) and evaporated to a syrup. Flash silica gelcolumn chromatography using 2-3% gradient of methanol in ethyl acetateyielded 9 as a colorless foam (1.04 g, 63%). ¹H-NMR ^(δ) 7.42 (d,J_(6,5)=8.4, 1H, H6 Urd), 7.53-6.97 (m, 13H, aromatic), 6.12 (d,J_(1′,2′)=8.0, 1H, H-1′), 5.41 (m, 3H, H5 Urd, H4NAcGal), 5.15 (dd,J_(3,4)=3.6, J_(3,2)=11.2, 2H, H₃NAcGal), 4.87 (dd, J_(2′,3′)=5.6,J_(2′,1′)=8.0, 1H, H2′), 4.63 (d, J_(1,2)=8.0, 2H, H1 NAcGal), 4.42 (d,J_(3′,2′)=5.6, 1H, H3′), 4.29-4.04 (m, 9H, H4′, H₂NAcGal, H5 NacGal,CH₂), 3.95-3.82 (m, 8H, H₆NAcGal, 2×OMe), 3.62-3.42 (m, 4H, H5′,H₆NAcGal), 3.26 (m, 2H, CH₂), 2.40-1.97 (m, 28H, CH₂, Ac), 1.95-1.30 (m,50H, CH₂). MS/ESI⁻ m/z 1727.0 (M−H)⁻.

[0603]2′-(N-α,ε-bis-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose)-L-lysyl)amino-2′-deoxy-5′-O-4,4′-dimethoxytrityluridine 3′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (10).Conjugate 9 (0.87 g, 0.50 mmol) was dissolved in dry dichloromethane (10ml) under argon and diisopropylethylamine (0.36 ml, 2.07 mmol) and1-methylimidazole (21 μL, 0.26 mmol) were added. The solution was cooledto 0° C. and 2-cyanoethyl diisopropylchlorophosphoramidite (0.19 ml,0.85 mmol) was added. The reaction mixture was stirred at rt for 1 hour,than cooled to 0° C. and quenched with anhydrous ethanol (0.5 ml). Afterstirring for 10 min the solution was concentrated under reduced pressure(40° C.) and the residue dissolved in dichloromethane andchromatographed on the column of silica gel using hexanes-ethyl acetate1:1, followed by ethyl acetate and finally ethyl acetate-acetone 1:1 (1%triethylamine was added to solvents) to afford the phosphoramidite 10(680 mg, 69%). ³¹P-NMR δ 152.0 (s), 149.3 (s). MS/ESI⁻ m/z 1928.0(M−H)⁻.

[0604]N-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol(11).12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose7 (850 mg, 1.56 mmol) was dissolved in DMF (5 ml) and to the solutionN-hydroxysuccinimide (215 mg, 1.87 mmol) and 1,3-dicyclohexylcarbodimide(386 mg, 1.87 mmol) were added. The reaction mixture was stirred at rtovernight, the precipitate was filtered off and to the filtrateD-threoninol (197 mg, 1.87 mmol) was added. The mixture was stirred atrt overnight and concentrated in vacuo. The residue was partitionedbetween dichloromethane and 5% NaHCO₃, the organic layer was washed withbrine, dried (Na₂SO₄) and evaporated to a syrup. Silica gel columnchromatography using 1-10% gradient of methanol in dichloromethaneafforded 11 as a colorless oil (0.7 g, 71%). ¹H-NMR δ 6.35 (d, J=7.6,1H, NH), 5.77 (d, J=8.0, 1H, NH), 5.44 (d, ^(J) _(4,3)=3.6, 1H, H4),5.37 (dd, ^(J) _(3,4)=3.6, ^(J) _(3,2)=11.2, 1H, H3), 4.77 (d, ^(J)_(1,2)=8.0, 1H, H1), 4.28-4.18 (m, 3H, CH₂, CH), 4.07-3.87 (m, 6H), 3.55(m, 1H, H5), 3.09 (d, J=3.2, 1H, OH), 3.02 (t, J=4.6, 1H, OH), 2.34 (t,J=7.4 2H, CH₂), 2.23 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.04 (s, 3H, Ac),1.76-1.61 (m, 2×CH₂), 1.35 (m, 14H, 7×CH₂), 1.29 (d, J=6.4, 3H, CH₃).MS/ESI⁻ m/z (M−H)⁻.

[0605]1-O-(4-Monomethoxytrityl)-N-(12′-hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol(12). To the solution of 11 (680 mg, 1.1 mmol) in dry pyridine (10 ml)p-anisylchlorotriphenylmethane (430 mg, 1.39 mmol) was added and therection mixture was stirred, protected from moisture, overnight.Methanol (3 ml) was added and the solution stirred for 15 min andevaporated in vacuo. The residue was partitioned between dichloromethaneand 5% NaHCO₃, the organic layer was washed with brine, dried (Na2SO₄)and evaporated to a syrup. Silica gel column chromatography using 1-3%gradient of methanol in dichloromethane afforded 12 as a white foam(0.75 g, 77%). ¹H-NMR δ 7.48-6.92 (m, 14H, aromatic), 6.15 (d, J=8.8,1H, NH), 5.56 (d, J=8.0, 1H, NH), 5.45 (d, ^(J) _(4,3)=3.2, 1H, H4),5.40 (dd, ^(J) _(3,4)=3.2, ^(J) _(3,2)=11.2, 1H, H3), 4.80 (d, ^(J)_(1,2)=8.0, 1H, H1), 4.3-4.13 (m, 3H, CH₂, CH), 4.25-3.92 (m, 4H, H6,H2, CH), 3.89 (s, 3H, OMe), 3.54 (m, 2H, H5, CH), 3.36 (dd, J=3.4,J=9.8, 1H, CH), 3.12 (d, J=2.8, 1H, OH), 2.31 (t, J=7.6, 2H, CH₂), 2.22(s, 3H, Ac), 2.13 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.80-1.55 (m, 2×CH₂),1.37 (m, 14H, 7×CH₂), 1.21 (d, J=6.4, 3H, CH₃). MS/ESI⁻ m/z 903.5(M−H)⁻.

[0606]1-O-(4-Monomethoxytrityl)-N-(12′-hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol3-O-(2-cyanoethyl N,N-diisopropylphosphorami-dite) (13). Conjugate 12(1.2 g, 1.33 mmol) was dissolved in dry dichloromethane (15 ml) underargon and diisopropylethylamine (0.94 ml, 5.40 mmol) and1-methylimidazole (55 μL, 0.69 mmol) were added. The solution was cooledto 0° C. and 2-cyanoethyl N,N-diisopropyl-chlorophosphoramidite (0.51ml, 2.29 mmol) was added. The reaction mixture was stirred at rt for 2hours, than cooled to 0° C. and quenched with anhydrous ethanol (0.5ml). After stirring for 10 min. the solution was concentrated underreduced pressure (40° C.) and the residue dissolved in dichloromethaneand chromatographed on the column of silica gel using 50-80% gradient ofethyl acetate in hexanes (1% triethylamine) to afford thephosphoramidite 13 (1.2 g, 82%). ³¹P-NMR δ 149.41 (s), 149.23 (s).

[0607] Oligonucleotide Synthesis

[0608] Phosphoramidites 10, and 13, were used along with standard2′-O-TBDMS and 2′-O-methyl nucleoside phosphoramidites. Synthesis wereconducted on a 394 (ABI) synthesizer using modified 2.5 μmol scaleprotocol with a 5 min coupling step for 2′-O-TBDMS protected nucleotidesand 2.5 min coupling step for 2′-O-methyl nucleosides. Couplingefficiency for the phosphoramidite 10 was lower than 50% while couplingefficiencies for phosphoramidite 13 was typically greater than 95% basedon the measurement of released trityl cations. Once the synthesis wascompleted, the oligonucleotides were deprotected. The 5′-trityl groupswere left attached to the oligomers to assist purification. Cleavagefrom the solid support and the removal of the protecting groups wasperformed as described herein with the exception of using 20% piperidinein DMF for 15 min for the removal of Fm protection prior methylaminetreatment. The 5′-tritylated oligomers were separated from shorter(trityl-off) failure sequences using a short column of SEP-PAK C-18adsorbent. The bound, tritylated oligomers were detritylated on thecolumn by treatment with 1% trifluoroacetic acid, neutralized withtriethylammonium acetate buffer, and than eluted. Further purificationwas achieved by reverse-phase HPLC. An example of aN-acetyl-D-galactosamine conjugate that can be synthesized usingphosphoramidite 13 is shown in FIG. 15.

[0609] Structures of the ribozyme conjugates were confirmed by MALDI-TOFMS.

[0610] Monomer Synthesis

[0611] 2′-Amino-2′-deoxyuridine-N-acetyl-D-galactosamine conjugate. Thebis-Fmoc protected lysine linker was attached to the 2′-amino group of2′-amino-2′-deoxyuridine using the EEDQ catalyzed peptide coupling. The5′-OH was protected with 4,4′-dimethoxytrityl group to give 1, followedby the cleavage of N-Fmoc groups with diethylamine to afford synthon 2in the high overall yield.

[0612] 2-acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactopyranose 4 wassynthesized with minor modifications according to the reported procedure(Findeis supra). Mercury salt catalyzed glycosylation of 4 with thebenzyl ester of 12-hydroxydodecanoic acid 5 afforded glycoside 6 in 81%yield. Hydrogenolysis of benzyl protecting group yielded 7 in aquantitative yield. The coupling of the sugar derivative with thenucleoside synthon was achieved through preactivation of the carboxylicfunction of 7 as N-hydroxysuccinimide ester 8, followed by coupling tolysyl-2′-aminouridine conjugate 2. The final conjugate 9 was thanphosphitylated under standard conditions to afford the phosphoramidite10 in 69% yield.

[0613] D-Threoninol-N-acetyl-D-galactosamine conjugate Using the similarstrategy as described above, D-threoninol was coupled to 7 to affordconjugate 11 in a good yield. Monomethoxytritylation, followed byphosphitylation yielded the desired phosphoramidite 13.

EXAMPLE 2 Synthesis of Oxime Linked Nucleic Acid/Peptide Conjugates(FIGS. 16 and 17)

[0614] 12-Hydroxydodecanoic acid benzyl ester Benzyl bromide (10.28 ml,86.45 mmol) was added dropwise to a solution of 12-hydroxydodecanoicacid (17 g, 78.59 mmol) and DBU (12.93 ml, 86.45 mmol) in absolute DMF(120 ml) under vigorous stirring at 0° C. After completeion of theaddition reaction mixture was warmed to a room temperature and leftovernight under stirring. TLC (hexane-ethylacetate 3:1) indicatedcomplete transformation of the starting material. DMF was removed underreduced pressure and the residue was partitioned between ethyl ether and1N HCl. Organic phase was separated, washed with saturated aq sodiumbicarbonate and dried over sodium sulfate. Sodium sulfate was filteredoff, filtrate was evaporated to dryness. The residue was crystallizedfrom hexane to give 21.15 g (92%) of the title compound as a whitepowder.

[0615] 12-O-N-Phthaloyl-dodecanoic acid benzyl ester (15).Diethylazodicarboxylate (DEAD, 16.96 ml, 107.7 mmol) was added dropwiseto the mixture of 12-Hydroxydodecanoic acid benzyl ester (21 g, 71.8mmol), triphenylphosphine (28.29 g, 107.7 mmol) and N-hydroxyphthalimide(12.88 g, 78.98 mmol) in absolute THF (250 ml) at −20°-−30° C. understirring. The reaction mixture was stirred at this temperature foradditional 2-3 h, after which time TLC (hexane-ethylacetate 3:1)indicated reaction completion. The solvent was removed in vacuo and theresidue was treated ether (250 ml). Formed precipitate oftriphenylphosphine oxide was filtered off, mother liquor was evaporatedto dryness and the residue was dissolved in methylene chloride andpurified by flash chromatography on silica gel in hexane-ethyl acetate(7:3). Appropriate fractions were pooled and evaporated to dryness toafford 26.5 g (84.4%) of compound 15.

[0616] 12-O-N-Phthaloyl-dodecanoic acid (16). Compound 15 (26.2 g, 59.9mmol) was dissolved in 225 ml of ethanol-ethylacetate (3.5:1) mixtureand 10% Pd/C (2.6 g) was added. The reaction mixture was hydrogenated inParr apparatus for 3 hours. Reaction mixture was filtered through celiteand evaporated to dryness. The residue was crystallized from methanol toprovide 15.64 g (75%) of compound 16.

[0617] 12-O-N-Phthaloyl-dodecanoic acid 2,3-di-hydroxy-propylamide (18)The mixture of compound 16 (15.03 g, 44.04 mmol),dicyclohexylcarbodiimide (10.9 g, 52.85 mmol) and N-hydroxysuccinimide(6.08 g, 52.85 mmol) in absolute DMF (150 ml) was stirred at roomtemperature overnight. TLC (methylene chloride-methanol 9:1) indicatedcomplete conversion of the starting material and formation of NHS ester17. Then aminopropanediol (4.01 g, 44 mmol) was added and the reactionmixture was stirred at room temperature for another 2 h. The formedprecipitate of dicyclohexylurea was removed by filtration, filtrate wasevaporated under reduced pressure. The residue was partitioned betweenethyl acetate and saturated aq sodium bicarbonate. The whole mixture wasfiltered to remove any insoluble material and clear layers wereseparated. Organic phase was concentrated in vacuo until formation ofcrystalline material. The precipitate was filtered off and washed withcold ethylacetate to produce 10.86 g of compound 17. Combined motherliquor and washings were evaporated to dryness and crystallized fromethylacetate to afford 3.21 g of compound 18. Combined yield—14.07 g(73.5%).

[0618] 12-O-N-Phthaloyl-dodecanoic acid 2-hydroxy,3-dimethoxytrityloxy-propylamide (19)_Dimethoxytrityl chloride (12.07 g,35.62 mmol) was added to a stirred solution of compound 18 (14.07 g,32.38 mmol) in absolute pyridine (130 ml) at 0° C. The reaction solutionwas kept at 0° C. overnight. Then it was quenched with MeOH (10 ml) andevaporated to dryness. The residue was dissolved in methylene chlorideand washed with saturated aq sodium bicarbonate. Organic phase wasseparated, dried over sodium sulfate and evaporated to dryness. Theresidue was purified by flash chromatography on silica gel using stepgradient of acetone in hexanes (3:7 to 1:1) as an eluent. Appropriatefractions were pooled and evaporated to provide 14.73 g (62%) ofcompound 19, as a colorless oil.

[0619] 12-O-N-Phthaloyl-dodecanoic acid2-O-(cyanoethyl-N.N-diisopropylamino-phosphoramidite),3-dimethoxytrityloxy-propylamide(20). Phosphitylated according to Sanghvi, et al., 2000, Organic ProcessResearch and Development, 4, 175-81.

[0620] Purified by flash chromatography on silica gel using stepgradient of acetone in hexanes (1:4 to 3:7) containing 0.5% oftriethylamine. Yield —82%, colourless oil.

[0621] Oxidation of Peptides

[0622] Peptide (3.3 mg, 3.3 μmol) was dissolved in 10 mM AcONa and 2 eqof sodium periodate (100 mM soln in water) was added. Final reactionvolume—0.5 ml. After 10 minutes reaction mixture was purified usinganalytical HPLC on Phenomenex Jupiter 5u C18 300A (150×4.6 mm) column;solvent A: 50 mM KH₂PO₄ (pH 3); solvent B: 30% of solvent A in MeCN;gradient B over 30 min. Appropriate fractions were pooled andconcentrated on a SpeedVac to dryness. Yield: quantitative.

[0623] Conjugation Reaction of Herzyme-ONH2-Linker with N-glyoxylPeptide (FIG. 17)

[0624] Herzyme (SEQ ID NO: 13) with a 5′-terminal linker (1000D) wasmixed with oxidized peptide (3-5 eq) in 50 mM KH2PO4 (pH3, reactionvolume 1 ml) and kept at room temperature for 24-48h. The reactionmixture was purified using analytical HPLC on a Phenomenex Jupiter 5uC18 300A (150×4.6 mm) column; solvent A: 10 mM TEAA; solvent B: 10 mMTEAA/MeCN. Appropriate fractions were pooled and concentrated on aSpeedVac to dryness to provide desired conjugate. ESMS: calculated:12699, determined: 12698.

EXAMPLE 5 Synthesis of Phospholipid Enzymatic Nucleic Acid Conjugates(FIG. 19)

[0625] A phospholipid enzymatic nucleic acid conjugate (see FIG. 19) wasprepared by coupling a C18H37 phosphoramidite to the 5′-end of anenzymatic nucleic acid molecule (Angiozyme™, SEQ ID NO: 24) during solidphase oligonucleotide synthesis on an ABI 394 synthesizer using standardsynthesis chemistry. A 5′-terminal linker comprising3′-AdT-di-Glycerol-5′, where A is Adenosine, dT is 2′-deoxy Thymidine,and di-Glycerol is a di-DMT-Glycerol linker (Chemgenes CAT numberCLP-5215), is used to attach two C18H37 phosphoramidites to theenzymatic nucleic acid molecule using standard synthesis chemistry.Additional equivalents of the C18H37 phosphoramidite were used for thebis-coupling. Similarly, other nucleic acid conjugates as shown in FIG.18 can be prepared according to similar methodology.

EXAMPLE 6 Synthesis of PEG Enzymatic Nucleic Acid Conjugates (FIG. 20)

[0626] A 40K-PEG enzymatic nucleic acid conjugate (see FIG. 20) wasprepared by post synthetic N-hydroxysuccinimide ester coupling of a PEGderivative (Shearwater Polymers Inc, CAT number PEG2-NHS) to the 5′-endof an enzymatic nucleic acid molecule (Angiozyme™, SEQ ID NO: 24). A5′-terminal linker comprising 3′-AdT-C6-amine-5′, where A is Adenosine,dT-C6-amine is 2′-deoxy Thymidine with a C5 linked six carbon aminelinker (Glen Research CAT number 10-1039-05), is used to attach the PEGderivative to the enzymatic nucleic acid molecule using NHS couplingchemistry.

[0627] Angiozyme™ with the C6dT-NH2 at the 5′ end was synthesized anddeprotected using standard oligonucleotide synthesis procedures asdescribed herein. The crude sample was subsequently loaded onto areverse phase column and rinsed with sodium chloride solution (0.5 M).The sample was then desalted with water on the column until theconcentration of sodium chloride was close to zero. Acetonitrile wasused to elute the sample from the column. The crude product was thenconcentrated and lyophilized to dryness.

[0628] The crude material (Angiozyme™) with 5′-amino linker (50 mg) wasdissolved in sodium borate buffer (1.0 mL, pH 9.0). The PEG NHS ester(200 mg) was dissolved in anhydrous DMF (1.0 mL). The Angiozyme™ buffersolution was then added to the PEG NHS ester solution. The mixture wasimmediately vortexed for 5 minutes. Sodium acetate buffer solution (5mL, pH 5.2) was used to quench the reaction. Conjugated material wasthen purified by ion-exchange and reverse phase chromatography.

EXAMPLE 7 Phamacokinetics of PEG Ribozyme Acid Conjugate (FIG. 21)

[0629] Forty-eight female C57B1/6 mice were given a single subcutaneous(SC) bolus of 30 mg/kg Angiozyme™ and 30 mg/kg Angiozyme™/40K PEGconjugate. Plasma was collected out to 24 hours post ribozyme injection.Plasma samples were analyzed for full length ribozyme by a hybridizationassay.

[0630] Oligonucleotides complimentary to the 5′ and 3′ ends ofAngiozyme™ were synthesized with biotin at one oligo, and FITC on theother oligo. A biotin oligo and FITC labeled oligo pair are incubated at1 ug/ml with known concentrations of Angiozyme™ at 75 degrees C. for 5min. After 10 minutes at RT, the mixture is allowed to bind tostreptavidin coated wells of a 96-wll plate for two hours. The plate iswashed with Tris-saline and detergent, and peroxidase labeled anti-FITCantibody is added. After one hour, the wells are washed, and theenzymatic reaction is developed, then read on an ELISA plate reader.Results are shown in FIG. 21.

EXAMPLE 8 Phamacokinetics of Phospholipid Ribozyme Conjugate (FIG. 22)

[0631] Seventy-two female C57B1/6 mice were given a single intravenous(4) bolus of 30 mg/kg Angiozyme™ and 30 mg/kg Angiozyme™ conjugated withphospholipid (FIG. 19). Plasma was collected out to 3 hours postribozyme injection. Plasma samples were analyzed for full lengthribozyme by a hybridization assay.

[0632] Oligonucleotides complimentary to the 5′ and 3′ ends ofAngiozyme™ were synthesized with biotin at one oligo, and FITC on theother oligo. A biotin oligo and FITC labeled oligo pair are incubated at1 ug/ml with known concentrations of Angiozyme™ at 75 degrees C. for 5min. After 10 minutes at RT, the mixture is allowed to bind tostreptavidin coated wells of a 96-wll plate for two hours. The plate iswashed with Tris-saline and detergent, and peroxidase labeled anti-FITCantibody is added. After one hr, the wells are washed, and the enzymaticreaction is developed, then read on an ELISA plate reader. Results areshown in FIG. 22.

EXAMPLE 9 Synthesis of Protein or Peptide Conjugates with BiodegradableLinkers (FIGS. 24-26, and 29)

[0633] Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using oxime and morpholino linkages. For example, atherapeutic antibody can be conjugated with PEG to improve the FIG. 24shows a non-limiting example of a synthetic approach for synthesizingpeptide or protein conjugates to PEG utilizing a biodegradable linker,the example shown is for a protein conjugate. Other conjugates can besynthesized in a similar manner where the protein or peptide isconjugated to molecules other than PEG, such as small molecules, toxins,radioisotopes, peptides or other proteins. (a) The protein of interest,such as an antibody or interferon, is synthesized with a terminal Serineor Threonine moiety that is oxidized, for example with sodium periodate.The oxidized protein is then coupled to a nucleic acid linker moleculethat is designed to be biodegradable, for example acytidine-deoxythymidine, cytidine-deoxyuridine,adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that containsan oxyamino (O—NH₂) function. Other biodegradable nucleic acid linkerscan be similarly used, for example other dimers, trimers, tetramers etc.that are designed to be biodegradable. The example shown makes use of a5′-oxyamino moiety, however, other examples can utilize an oxyamino atother positions within the nucleic acid molecule, for example at the2′-position, 3′-position, or at a nucleic acid base position. (b) Theprotein/nucleic acid conjugate is then oxidized to generate a dialdehydefunction that is coupled to PEG molecule comprising an amino group(H₂N-PEG), for example a PEG molecule with an amino linker. Other aminocontaining molecules can be conjugated as shown in the figure, forexample small molecules, toxins, or radioisotope labeled molecules.

[0634] Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using oxime and phosphoramidate linkages. FIG. 25 shows anon-limiting example of a synthetic approach for synthesizing peptide orprotein conjugates to PEG utilizing a biodegradable linker, the exampleshown is for a protein conjugate. Other conjugates can be synthesized ina similar manner where the protein or peptide is conjugated to moleculesother than PEG, such as small molecules, toxins, radioisotopes, peptidesor other proteins. The protein of interest, such as an antibody orinterferon, is synthesized with a terminal Serine or Threonine moietythat is oxidized, for example with sodium periodate. The oxidizedprotein is then coupled to a nucleic acid linker molecule that isdesigned to be biodegradable, for example a cytidine-deoxythymidine,cytidine-deoxyuridine, adenosine-deoxythymidine, oradenosine-deoxyuridine dimer that contains an oxyamino (O—NH₂) functionand a terminal phosphate group. Terminal phosphate groups can beintroduced during synthesis of the nucleic acid molecule using chemicalphosphorylation reagents, such as Glen Research Cat Nos. 10-1909-02,10-1913-02, 10-1914-02, and 10-1918-02. Other biodegradable nucleic acidlinkers can be similarly used, for example other dimers, trimers,tetramers etc. that are designed to be biodegradable. The example shownmakes use of a 5′-oxyamino moiety, however, other examples can utilizean oxyamino at other positions within the nucleic acid molecule, forexample at the 2′-position, 3′-position, or at a nucleic acid baseposition. The protein/nucleic acid conjugate terminal phosphate group isthen activated with an activator reagent, such as NMI and/or tetrazole,and coupled a PEG molecule comprising an amino group (H₂N-PEG), forexample a PEG molecule with an amino linker. Other amino containingmolecules can be conjugated as shown in the figure, for example smallmolecules, toxins, or radioisotope labeled molecules.

[0635] Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using phosphoramidate linkages. FIG. 26 shows a non-limitingexample of a synthetic approach for synthesizing peptide or proteinconjugates to PEG utilizing a biodegradable linker, the example shown isfor a protein conjugate. Other conjugates can be synthesized in asimilar manner where the protein or peptide is conjugated to moleculesother than PEG, such as small molecules, toxins, radioisotopes, peptidesor other proteins. (a) A nucleic acid linker molecule that is designedto be biodegradable, for example a cytidine-deoxythymidine,cytidine-deoxyuridine, adenosine-deoxythymidine, oradenosine-deoxyuridine dimer, is synthesized with a terminal phosphategroup. Other biodegradable nucleic acid linkers can be similarly used,for example other dimers, trimers, tetramers etc. that are designed tobe biodegradable. The protein/nucleic acid conjugate terminal phosphategroup is then activated with an activator reagent, such as NMI and/ortetrazole, and coupled a PEG molecule comprising an amino group(H₂N-PEG), for example a PEG molecule with an amino linker. Other aminocontaining molecules can be conjugated as shown in the figure, forexample small molecules, toxins, or radioisotope labeled molecules. Theterminal protecting group, for example a dimethoxytrityl group, isremoved from the conjugate and a terminal phosphite group is introducedwith a phosphitylating reagent, such as N,N-diisopropyl-2-cyanoethylchlorophosphoramidite. (b) The PEG/nucleic acid conjugate is thencoupled to a peptide or protein comprising an amino group, such as theamino terminus or amino side chain of a suitably protected peptide orprotein or via an amino linker. The conjugate is then oxidized and anyprotecting groups are removed to yield the protein/PEG conjugatecomprising a biodegradable linker.

[0636] Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using phosphoramidate linkages from coupling protein-basedphosphoramidites. FIG. 29 shows a non-limiting example of a syntheticapproach for synthesizing peptide or protein conjugates to PEG utilizinga biodegradable linker, the example shown is for a protein conjugate.Other conjugates can be synthesized in a similar manner where theprotein or peptide is conjugated to molecules other than PEG, such assmall molecules, toxins, radioisotopes, peptides or other proteins. Theprotein of interest, such as an antibody or interferon, is synthesizedwith a terminal Serine, Threonin, or Tyrosine moiety that isphosphitylated, for example with N,N-diisopropyl-2-cyanoethylchlorophosphoramidite. The phosphitylated protein is then coupled to anucleic acid linker molecule that is designed to be biodegradable, forexample a cytidine-deoxythymidine, cytidine-deoxyuridine,adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that containsconjugated PEG molecule as described in FIG. 18. Other biodegradablenucleic acid linkers can be similarly used, for example other dimers,trimers, tetramers etc. that are designed to be biodegradable.

EXAMPLE 10 Galactosamine Ribozyme Conjugate Targeting HBV

[0637] A nuclease-resistance ribozyme directed against the Heptatitis Bviral RNA (HBV) (HepBzyme™) is in early stages of preclinicaldevelopment. HepBzyme, which targets site 273 of the Hepatitis B viralRNA, has produced statistically significant decreases in serum HBVlevels in a HBV transgenic mouse model in a dose-dependent manner (30and 100 mg/kg/day). In an effort to improve hepatic uptake by targetingthe asialoglycoprotein receptor, a series of 5 branched galactosamineresidues were attached via phosphate linkages to the 5′-terminus ofHepBzyme (Gal-HepBzyme). The affect of the galactosamine conjugation onHepBzyme was assessed by quantitation of ³²P-labeled HepBzyme andGal-HepBzyme in plasma, liver and kidney of mice following a single SCbolus administration of 30 mg/kg. The plasma disposition of the intactribozyme was similar between Gal-HepBzyme and HepBzyme. An approximatethree-fold increase in the maximum observed concentration of intactribozyme in liver (C_(max)) was observed in liver for Gal-HepBzyme(6.1±1.8 ng/mg) vs. HepBzyme (2.2±0.8 ng/mg) (p<0.05). The area underthe curve (AUCall) for Gal-HepBzyme was also increased by approximatelytwo-fold. This was accompanied by a substantial decrease (approximately40%) in the AUCall for intact ribozyme in kidney. In addition to thesignificant increase in C_(max) observed for intact Gal-HepBzyme in theliver, there was an increase in the total number of ribozymeequivalents, which may be suggestive of increased affinity of both theintact ribozyme and metabolites for asialoglycoprotein receptor andgalactose-specific receptors in the liver. These data demonstrate thatconjugation of a ribozyme with galactosamine produces a compound with amore favorable disposition profile, and illustrates the utility ofconjugated ribozymes with improved in vivo pharmacokinetics andbiodistribution.

EXAMPLE 11 Synthesis of siNA Conjugates

[0638] siNA molecules can be designed to interact with various sites ina target RNA message, for example, target sequences within the RNAsequence. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences. The siNA molecules can bechemically synthesized using methods described herein. Inactive siNAmolecules that are used as control sequences can be synthesized byscrambling the sequence of the siNA molecules such that it is notcomplementary to the target sequence. Generally, siNA constructs can bysynthesized using solid phase oligonucleotide synthesis methods asdescribed herein (see for example Usman et al., U.S. Pat. Nos.5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323;6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086;6,008,400; 6,111,086 all incorporated by reference herein in theirentirety).

[0639] In a non-limiting example, RNA oligonucleotides are synthesizedin a stepwise fashion using the phosphoramidite chemistry as is known inthe art. Standard phosphoramidite chemistry involves the use ofnucleosides comprising any of 5′-O-dimethoxytrityl,2′-O-tert-butyldimethylsilyl, 3′-O-2-CyanoethylN,N-diisopropylphosphoroamidite groups, and exocyclic amine protectinggroups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyrylguanosine). Alternately, 2′-O-Silyl Ethers can be used in conjunctionwith acid-labile 2′-O-orthoester protecting groups in the synthesis ofRNA as described by Scaringe supra. Differing 2′ chemistries can requiredifferent protecting groups, for example 2′-deoxy-2′-amino nucleosidescan utilize N-phthaloyl protection as described by Usman et al., U.S.Pat. No. 5,631,360, incorporated by reference herein in its entirety).

[0640] During solid phase synthesis, each nucleotide is addedsequentially (3′- to 5′-direction) to the solid support-boundoligonucleotide. The first nucleoside at the 3′-end of the chain iscovalently attached to a solid support (e.g., controlled pore glass orpolystyrene) using various linkers. The nucleotide precursor, aribonucleoside phosphoramidite, and activator are combined resulting inthe coupling of the second nucleoside phosphoramidite onto the 5′-end ofthe first nucleoside. The support is then washed and any unreacted5′-hydroxyl groups are capped with a capping reagent such as aceticanhydride to yield inactive 5′-acetyl moieties. The trivalent phosphoruslinkage is then oxidized to a more stable phosphate linkage. At the endof the nucleotide addition cycle, the 5′-O-protecting group is cleavedunder suitable conditions (e.g., acidic conditions for trityl-basedgroups and Fluoride for silyl-based groups). The cycle is repeated foreach subsequent nucleotide.

[0641] Modification of synthesis conditions can be used to optimizecoupling efficiency, for example by using differing coupling times,differing reagent/phosphoramidite concentrations, differing contacttimes, differing solid supports and solid support linker chemistriesdepending on the particular chemical composition of the siNA to besynthesized. Deprotection and purification of the siNA can be performedas is generally described in Deprotection and purification of the siNAcan be performed as is generally described in Usman et al., U.S. Pat.No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, andBellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S.Pat. No. 6,303,773, or Scaringe supra, incorporated by reference hereinin their entireties. Additionally, deprotection conditions can bemodified to provide the best possible yield and purity of siNAconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

[0642] The introduction of conjugate moieties is accomplished eitherduring solid phase synthesis using phosphoramidite chemistry describedabove, or post-synthetically using, for example, N-hydroxysuccinimide(NHS) ester coupling to an amino linker present in the siNA. Typically,a conjugate introduced during solid phase synthesis will be added to the5′-end of a nucleic acid sequence as the final coupling reaction in thesynthesis cycle using the phosphoramidite approach. Coupling conditionscan be optimized for high yield coupling, for example by modification ofcoupling times and reagent concentrations to effectuate efficientcoupling. As such, the 5′-end of the sense strand of a siNA molecule isreadily conjugated with a conjugate moiety having a reactive phosphorusgroup available for coupling (e.g., a compound having Formulae 1, 5, 8,55, 56, 57, 60, 86, 92, 104, 110, 113, 115, 116, 117, 118, 120, or 122)using the phosphoramidite approach, providing a 5′-terminal conjugate(see for example FIG. 41).

[0643] Conjugate precursors having a reactive phosphorus group and aprotected hydroxyl group can be used to incorporate a conjugate moietyanywhere in the siNA sequence, such as in the loop portion of a singlestranded hairpin siNA construct (see for example FIG. 42). For example,using the phosphoramidite approach, a conjugate moiety comprising aphosphoramidite and protected hydroxyl (e.g., a compound having Formulae86, 92, 104, 113, 115, 116, 117, 118, 120, or 122 herein) is firstcoupled at the desired position within the siNA sequence using solidphase synthesis phosphoramidite coupling. Second, removal of theprotecting group (e.g., dimethoxytrityl) allows coupling of additionalnucleotides to the siNA sequence. This approach allows the conjugatemoiety to be positioned anywhere within the siNA molecule.

[0644] Conjugate derivatives can also be introduced to a siNA moleculepost synthetically. Post synthetic conjugation allows a conjugate moietyto be introduced at any position within the siNA molecule where anappropriate functional group is present (e.g., a C5 alkylamine linkerpresent on a nucleotide base or a 2′-alkylamine linker present on anucleotide sugar can provide a point of attachment for an NHS-conjugatemoiety). Generally, a reactive chemical group present in the siNAmolecule is unmasked following synthesis, thus allowing post-syntheticcoupling of the conjugate to occur. In a non-limiting example, anprotected amino linker containing nucleotide (e.g., TFA protected C5propylamino thymidine) is introduced at a desired position of the siNAduring solid phase synthesis. Following cleavage and deprotection of thesiNA, the free amine is made available for NHS ester coupling of theconjugate at the desired position within the siNA sequence, such as atthe 3′-end of the sense and/or antisense strands, the 3′ and/or 5′-endof the sense strand, or within the siNA sequence, such as in the loopportion of a single stranded hairpin siNA sequence.

[0645] A conjugate moiety can be introduced at different locationswithin a siNA molecule using both solid phase synthesis andpost-synthetic coupling approaches. For example, solid phase synthesiscan be used to introduce a conjugate moiety at the 5′-end of the siNA(e.g. sense strand) and post-synthetic coupling can be used to introducea conjugate moiety at the 3′-end of the siNA (e.g. sense strand and/orantisense strand). As such, a siNA sense strand having 3′ and 5′ endconjugates can be synthesized (see for example FIG. 41). Conjugatemoieties can also be introduced in other combinations, such as at the5′-end, 3′-end and/or loop portions of a siNA molecule (see for exampleFIG. 42).

EXAMPLE 12 Phamacokinetics of siNA Conjugates (FIG. 43)

[0646] Three nuclease resistant siNA molecule targeting site 1580 ofhepatitis B virus (HBV) RNA were designed using Stab 7/8 chemistry (seeTable IV) and a 5′-terminal conjugate moiety.

[0647] One siNA conjugate comprises a branched cholesterol conjugatelinked to the sense strand of the siNA. The “cholesterol” siNA conjugatemolecule has the structure shown below:

[0648] where T stands for thymidine, B stands for inverted deoxyabasic,G stands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, Gstands for 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, ustands for 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a standsfor adenosine, and s stands for phosphorothioate linkage.

[0649] Another siNA conjugate comprises a branched phospholipidconjugate linked to the sense strand of the siNA. The “phospholipid”siNA conjugate molecule has the structure shown below:

[0650] where T stands for thymidine, B stands for inverted deoxyabasic,G stands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, Gstands for 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, ustands for 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a standsfor adenosine, and s stands for phosphorothioate linkage.

[0651] Another siNA conjugate comprises a polyethylene glycol (PEG)conjugate linked to the sense strand of the siNA. The “PEG” siNAconjugate molecule has the structure shown below:

[0652] where T stands for thymidine, B stands for inverted deoxyabasic,G stands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, Gstands for 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, ustands for 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a standsfor adenosine, and s stands for phosphorothioate linkage.

[0653] The Cholesterol, Phospholipid, and PEG conjugates were evaluatedfor pharmakokinetic properties in mice compared to a non-conjugated siNAconstruct having matched chemistry and sequence. This study wasconducted in female CD-1 mice approximately 26 g (6-7 weeks of age).Animals were housed in groups of 3. Food and water were provided adlibitum. Temperature and humidity were according to Pharmacology TestingFacility performance standards (SOP's) which are in accordance with the1996 Guide for the Care and Use of Laboratory Animals (NRC). Animalswere acclimated to the facility for at least 3 days prior toexperimentation.

[0654] Absorbance at 260 nm was used to determine the actualconcentration of the stock solution of pre-annealed HBV siNA. Anappropriate amount of HBV siNA was diluted in sterile veterinary gradenormal saline (0.9%) based on the average body weight of the mice. Asmall amount of the antisense (Stab 7) strand was internally labeledwith gamma ³²P-ATP. The ³²P-labeled stock was combined with excess sensestrand (Stab 8) and annealed. Annealing was confirmed prior tocombination with unlabled drug. Each mouse received a subcutaneous bolusof 30 mg/kg (based on duplex) and approximately 10 million cpm (specificactivity of approximately 15 cpm/ng).

[0655] Three animals per timepoint (1, 4, 8, 24, 72, 96 h) wereeuthanized by CO2 inhalation followed immediately by exsanguination.Blood was sampled from the heart and collected in heparinized tubes.After exsanguination, animals were perfused with 10-15 mL of sterileveterinary grade saline via the heart. Samples of liver were thencollected and frozen.

[0656] Tissue samples were homogenized in a digestion buffer prior tocompound quantitation. Quantitation of intact compound was determined byscintillation counting followed by PAGE and phosphorimage analysis.Results are shown in FIG. 43. As shown in the figure, the conjugatedsiNA constructs shown vastly improved liver PK compared to theunconjugated siNA construct.

EXAMPLE 13 Cell culture of siNA Conjugates (FIG. 44)

[0657] The Cholesterol conjugates and Phospholipid conjugated siNAconstructs described in Example 12 above were evaluated for cell cultureefficacy in a HBV cell culture system.

[0658] Transfection of HepG2 Cells with psHBV-1 and siNA

[0659] The human hepatocellular carcinoma cell line Hep G2 was grown inDulbecco's modified Eagle media supplemented with 10% fetal calf serum,2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,25 mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. Togenerate a replication competent cDNA, prior to transfection the HBVgenomic sequences are excised from the bacterial plasmid sequencecontained in the psHBV-1 vector. Other methods known in the art can beused to generate a replication competent cDNA. This was done with anEcoRI and Hind III restriction digest. Following completion of thedigest, a ligation was performed under dilute conditions (20 μg/ml) tofavor intermolecular ligation. The total ligation mixture was thenconcentrated using Qiagen spin columns.

[0660] siNA Activity Screen and Dose Response Assay

[0661] Transfection of the human hepatocellular carcinoma cell line, HepG2, with replication-competent HBV DNA results in the expression of HBVproteins and the production of virions. To test the efficacy of siNAconjugates targeted against HBV RNA, the Cholesterol siNA conjugate andPhospholipid siNA conjugate described in Example 12 were compared to anon-conjugated control siNA (see FIG. 44). An inverted sequence duplexwas used as a negative control for the unconjugated siNA. Dose responsestudies were performed in which HBV genomic DNA was transfected with HBVgenomic DNA with lipid at 12.5 ug/ml into Hep G2 cells. 24 hours aftertransfection with HBV DNA, cell culture media was removed and siNAduplexes were added to cells without lipid at 10 uM, 5, uM, 2.5 uM, 1uM, and 100 nm and the subsequent levels of secreted HBV surface antigen(HBsAg) were analyzed by ELISA 72 hours post treatment (see FIG. 44). Todetermine siNA activity, HbsAg levels were measured followingtransfection with siNA. Immulon 4 (Dynax) microtiter wells were coatedovernight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1μg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5). Thewells were then washed 4× with PBST (PBS, 0.05% Tween® 20) and blockedfor 1 hr at 37° C. with PBST, 1% BSA. Following washing as above, thewells were dried at 37° C. for 30 min. Biotinylated goat ant-HBsAg(Accurate YVS1807) was diluted 1:1000 in PBST and incubated in the wellsfor 1 hr. at 37° C. The wells were washed 4× with PBST.Streptavidin/Alkaline Phosphatase Conjugate (Pierce 21324) was dilutedto 250 ng/ml in PBST, and incubated in the wells for 1 hr. at 37° C.After washing as above, p-nitrophenyl phosphate substrate (Pierce 37620)was added to the wells, which were then incubated for 1 hour at 37° C.The optical density at 405 nm was then determined. As shown in FIG. 44,the phospholipid and cholesterol conjugates demonstrate marked dosedependent inhibition of HBsAg expression compared to the unconjugatedsiNA construct when delivered to cells without any transfection agent(lipid).

EXAMPLE 14 Purification of Nucleic Acid Conjugates

[0662] Nucleic acid conjugates of the invention can be purified using,for example, anion exchange, reverse phase, and/or hydrophobicinteraction chromoatography. Non-lipophilic nucleic acid conjugates ofthe invention (e.g., PEG, PEI, polyamine conjugates) are readilypurified using anion exchange chromatography as is known in the art.Lipophilic nucleic acid conjugates of the invention (e.g., cholesterol,phospholipid, or alkyl polymer conjugates) can be purified using reversephase chromatography as is known in the art and/or by hydrophobicinteraction chromatography. Hydrophobic interaction chromotography (HIC)allows high efficiency purification of nucleic acid conjugates of theinvention without the use of organic solvents.

[0663] Hydrophobic interaction chromatography is based on interactionsbetween hydrophobic groups on the molecules to be purified or isolatedand the corresponding HIC resin. HIC utilizes such hydrophobicinteractions in highly polar non-denaturing buffers. Several differentHIC resins can be utilized in purifying hydrophobic polynucleotideconjugates (e.g., siNA conjugates). Examples of HIC resins include butare not limited to ether, phenyl, butyl, or hexyl stationary phases suchas Toyopearl.650 S Phenyl, TSK GEL Phenyl-5PW, TSK GEL Ether-5PW,Toyopearl Ether-650, Toyopearl Phenyl-650, Toyopearl Butyl-650, andToyopearl Hexyl-650 from TosoHaas and Fractogel EMD Phenyl S from Merck.Various conditions that can be altered to achieve highly purifiedmaterial using HIC include alterations in the concentration of salts inbuffers, use of different resins, varying pH and temperature.

[0664] In a non-limiting example, HIC was used to purify a Stab 7/8(Table IV) siNA cholesterol conjugate having SEQ ID NO: 24 (see forexample FIG. 30) and a Stab 7/8 (Table IV) siNA phospholipid conjugatehaving SEQ ID NO: 26 (see for example FIG. 19). The purification bufferreagents used in the HIC purification consisted of Ammonium Sulfate,Sodium Phosphate Monobasic and Dibasic (see Table V) which werepurchased from VWR. The material was purified on a Waters LC-2000preparative system including an LC Controller and pump and a waters24487 dual wavelength UV Detector. The system is controlled byMillennium software version 4. The buffers and loading material ispassed through a heat exchanger, such as a Timberline TL50D (TimberlineInstruments (Boulder, Colo.). The Columns used in the developmentincluded a Pharmacia HR 5/5, Pharmacia HR 16/10 and a Pharmacia Fineline35. The columns were packed with Toyopearl Phenyl-650s (TosohBioscience, LLC Montgomeryville, Pa.) to a bed height of 5 cm, 10 cm and10 cm respectively as the process was scaled up. Further work includedthe Toyopearl 650 S Phenyl to the Tosoh Bioscience TSK GEL Phenyl-5PWand the Fractogel EMD Phenyl S.

[0665] The deprotected cholesterol siNA and phospholipid siNA conjugateswere diluted in Milli-Q-water and ammonium sulfate was added to a 2Mconcentration following filtration and rinsing of the filter withMilli-Q-water. The addition of solid ammonium sulfate as a dry powderresulted in precipitation of the siNA conjugates. This process has beenfurther optimized so that the oligonucleotide is diluted inMilli-Q-water and then the volume is doubled with 2 M ammonium sulfateyielding a solution of 1 M ammonium sulfate with 10 mM Sodium Phosphate.

[0666] The purified conjugated siRNA is eluted from the column duringstep 2 of the gradient (see Table VI). This step also desalts themolecule. The eluate was collected as fractions, which were analyzed bySAX or RP HPLC to determine purity. Fractions containing the conjugatedproduct were pooled and this pool was analyzed by SAX or RP HPLC forpurity. The pooled material was stored 2-8° C. until annealed to thecomplementary strand and desalted.

[0667] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein are exemplary and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art, which are encompassedwithin the spirit of the invention, are defined by the scope of theclaims.

[0668] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications can be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. Thus, such additional embodiments are within the scope of thepresent invention and the following claims.

[0669] The invention illustratively described herein suitably can bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by various embodiments, optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by thedescription and the appended claims.

[0670] In addition, where features or aspects of the invention aredescribed in terms of Markush groups or other grouping of alternatives,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group or other group.

[0671] Other embodiments are within the following claims. TABLE ICharacteristics of naturally occurring ribozymes Group I Introns Size:˜150 to > 1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: aftack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintenance of the active structure. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies[^(i),^(ii)]. Complete kinetic framework established for one ribozyme[^(iii),^(iv),^(v),^(vi)]. Studies of ribozyme folding and substratedocking underway [^(vii),^(viii),^(ix)]. Chemical modificationinvestigation of important residues well established [^(xxi)]. The small(4-6 nt) binding site may make this ribozyme too non-specific fortargeted RNA cleavage, however, the Tetrahymena group I intron has beenused to repair a “defective” β-galactosidase message by the ligation ofnew β-galactosidase sequences onto the defective message [^(xii)] RNAseP RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portion of aubiquitous ribonucleoprotein enzyme. Cleaves tRNA precursors to formmature tRNA [^(xiii)] Reaction mechanism: possible attack by M²⁺-OH togenerate cleavage products with 3′-OH and 5′-phosphate. RNAse P is foundthroughout the prokaryotes and eukaryotes. The RNA subunit has beensequenced from bacteria, yeast, rodents, and primates. Recruitment ofendogenous RNAse P for therapeutic applications is possible throughhybridization of an External Guide Sequence (EGS) to the target RNA[^(xiv),^(xv)] Important phosphate and 2′ OH contacts recentlyidentified [^(xvi),^(xvii)] Group II Introns Size: > 1000 nucleotides.Trans cleavage of target RNAs recently demonstrated [^(xviii),^(xvix)].Sequence requirements not fully determined. Reaction mechanism: 2′-OH ofan internal adenosine generates cleavage products with 3′-OH and a“lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Only naturalribozyme with demonstrated participation in DNA cleavage [^(xx),^(xxi)]in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [^(xxii)].Important 2′ OH contacts beginning to be identified [^(xxiii)] Kineticframework under development [^(xxiv)] Neurospora VS RNA Size: ˜144nucleotides. Trans cleavage of hairpin target RNAs recently demonstrated[^(xxv)]. Sequence requirements not fully determined. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. Binding sites andstructural requirements not fully determined. Only 1 known member ofthis class. Found in Neurospora VS RNA. Hammerhead Ribozyme (see textfor references) Size: ˜13 to 40 nucleotides. Requires the targetsequence UH immediately 5′ of the cleavage site. Binds a variable numbernucleotides on both sides of the cleavage site. Reaction mechanism:attack by 2′-OH 5′ to the scissile bond to generate cleavage productswith 2′,3′-cyclic phosphate and 5′-OH ends. 14 known members of thisclass. Found in a number of plant pathogens (virusoids) that use RNA asthe infectious agent. Essential structural features largely defined,including 2 crystal structures [^(xxvi),^(xxvii)] Minimal ligationactivity demonstrated (for engineering through in vitro selection)[^(xxviii)] Complete kinetic framework established for two or moreribozymes [^(xxix)] Chemical modification investigation of importantresidues well established [^(xxx)]. Hairpin Ribozyme Size: ˜50nucleotides. Requires the target sequence GUC immediately 3′of thecleavage site. Binds 4-6 nucleotides at the 5′-side of the cleavage siteand a variable number to the 3′-side of the cleavage site. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. 3 known members ofthis class. Found in three plant pathogen (satellite RNAs of the tobaccoringspot virus, arabis mosaic virus and chicory yellow mottle virus)which uses RNA as the infectious agent. Essential structural featureslargely defined [^(xxxi),^(xxxii),^(xxiii),^(xxxiv)] Ligation activity(in addition to cleavage activity) makes ribozyme amenable toengineering through in vitro selection [^(xxxv)] Complete kineticframework established for one ribozyme [^(xxxvi)] Chemical modificationinvestigation of important residues begun [^(xxxvii),^(xxxviii)].Hepatitis Delta Virus (HDV) Ribozyme Size: ˜60 nucleotides. Transcleavage of target RNAs demonstrated [^(xxxix)]. Binding sites andstructural requirements not fully determined, although no sequences 5′of cleavage site are required. Folded ribozyme contains a pseudoknotstructure [^(x1)]. Reaction mechanism: attack by 2′-OH 5′ to thescissile bond to generate cleavage products with 2′,3′-cyclic phosphateand 5′-OH ends. Only 2 known members of this class. Found in human HDV.Circular form of HDV is active and shows increased nuclease stability[^(x1i)]

[0672] TABLE II Reagent Equivalents Amount Wait Time* DNA Wait Time*2′-O-methyl Wait Time *RNA A. 2.5 μmol Synthesis Cycle ABI 394Instrument Phosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-EthylTetrazole 23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL5 sec 5 sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 secBeaucage 12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NANA NA B. 0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 1531 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min465 sec Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124μL 5 sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 secIodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle96 well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* WaitTime* 2′-O- Wait Time* Reagent 2′-O-methyl/Ribo methyl/Ribo DNA methylRibo Phosphoramidites   22/33/66 40/60/120 μL 60 sec 180 sec 360 secS-Ethyl Tetrazole   70/105/210 40/60/120 μL 60 sec 180 min 360 secAcetic Anhydride  265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl 502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA  238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine  6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage   34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA

[0673] TABLE III Peptides for Conjugation SEQ ID Peptide Sequence NOANTENNAP RQI KIW FQN RRM KWK K amide 14 EDIA Kaposi AAV ALL PAV LLA LLAP + VQR 15 fibroblast KRQ KLMP growth factor caiman MGL GLH LLV LAA ALQGA 16 crocodylus Ig(5) light chain HIVenvelope GAL FLG FLG AAG STM GA +PKS 17 glycoprotein KRK 5 (NLS of the SV40) gp41 HIV-1 Tat RKK RRQ RRR18 Influenza GLFEAIAGFIENGWEGMIDGGGYC 19 hemagglutinin envelopglycoprotein RGD peptide X-RGD-X 20 where X is any amino acid or peptidetransportan A GWT LNS AGY LLG KIN LKA LAA 21 LAK KIL Somatostatin (S)FCYWK TCT 22 (tyr-3- octreotate) Pre-S-peptide (S)DH QLN PAF 23

[0674] TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at S/AS 3′-ends “Stab 1” Ribo Ribo — 5at 5′- S/AS end 1 at 3′- end “Stab 2” Ribo Ribo — All Usually ASlinkages “Stab 3” 2′-fluoro Ribo — 4 at 5′- Usually S end 4 at 3′- end“Stab 4” 2′-fluoro Ribo 5′ and — Usually S 3′-ends “Stab 5” 2′-fluoroRibo — 1 at 3′- Usually AS end “Stab 6” 2′-O-Methyl Ribo 5′ and —Usually S 3′-ends “Stab 7” 2′-fluoro 2′-deoxy 5′ and — Usually S 3′-ends“Stab 8” 2′-fluoro 2′-O- — 1 at 3′- Usually AS Methyl end “Stab 9” RiboRibo 5′ and — Usually S 3′-ends “Stab 10” Ribo Ribo — 1 at 3′- UsuallyAS end “Stab 11” 2′-fluoro 2′-deoxy - 1 at 3′- Usually AS end “Stab 12”2′-fluoro LNA 5′ and Usually S 3′-ends “Stab 13” 2′-fluoro LNA 1 at 3′-Usually AS end “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′- Usually AS end 1 at3′- end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′- Usually AS end 1 at 3′- end“Stab 16 Ribo 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 17”2′-O-Methyl 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 18” 2′-fluoro2′-O- 5′ and 1 at 3′- Usually S Methyl 3′-ends end “Stab 19” 2′-fluoro2′-O- 3′-end Usually AS Methyl “Stab 20” 2′-fluoro 2′-deoxy 3′-endUsually AS “Stab 21” 2′-fluoro Ribo 3′-end Usually AS “Stab 22” RiboRibo 3′-end Usually AS —

[0675] TABLE V Typical Hydrophobic Interaction Chromatography (HIC)Buffers pH Conductivity Equilibrium 1.0 M  10 mM Sodium 7.0 ± 0.3 (A)Ammonium Phosphate, Sulfate, Elution (B) N/A 100 mM Sodium 7.0 ± 0.3Phosphate, Elution 2 (C) N/A N/A N/A <20 μS/cm

[0676] TABLE VI Typical HIC Gradient Gradient Step Buffer Time Step 1100% A to 100% B 20 cv Step 2 100% B to 20% C 30 cv Step 3  20% C to100% C 30 cv

[0677]

1 24 1 10 RNA Artificial Sequence Description of Artificial SequenceExample of a Stem II region 1 gccguuaggc 10 2 15 RNA Artificial SequenceDescription of Artificial Sequence Generic Target Nucleic Acid 2nnnnnnunnn nnnnn 15 3 36 RNA Artificial Sequence Description ofArtificial Sequence Enzymatic Nucleic Acid 3 nnnnnnncug augagnnngaaannncgaaa nnnnnn 36 4 14 RNA Artificial Sequence Description ofArtificial Sequence Generic Target Nucleic Acid 4 nnnnncnnnn nnnn 14 535 RNA Artificial Sequence Description of Artificial Sequence EnzymaticNucleic Acid 5 nnnnnnncug augagnnnga aannncgaan nnnnn 35 6 15 RNAArtificial Sequence Description of Artificial Sequence Generic TargetNucleic Acid 6 nnnnnnngnn nnnnn 15 7 35 RNA Artificial SequenceDescription of Artificial Sequence Enzymatic Nucleic Acid 7 nnnnnnnugauggcaugcac uaugcgcgnn nnnnn 35 8 48 RNA Artificial Sequence Descriptionof Artificial Sequence Enzymatic Nucleic Acid 8 gugugcaacc ggaggaaacucccuucaagg acgaaagucc gggacggg 48 9 16 RNA Artificial SequenceDescription of Artificial Sequence Target Nucleic Acid 9 gccguggguugcacac 16 10 36 RNA Artificial Sequence Description of ArtificialSequence Enzymatic Nucleic Acid 10 gugccuggcc gaaaggcgag ugaggucugccgcgcn 36 11 15 RNA Artificial Sequence Description of ArtificialSequence Target Nucleic Acid 11 gcgcggcgca ggcac 15 12 16 DNA ArtificialSequence Description of Artificial Sequence Enzymatic Nucleic Acid Motif12 rggctagcta caacga 16 13 33 RNA Artificial Sequence Description ofArtificial Sequence Enzymatic Nucleic Acid 13 gcaguggccg aaaggcgagugaggucuagc uca 33 14 16 PRT Artificial Sequence misc_feature Syntheticpeptide 14 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp LysLys 1 5 10 15 15 26 PRT Artificial Sequence misc_feature Syntheticpeptide 15 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu AlaPro 1 5 10 15 Val Gln Arg Lys Arg Gln Lys Leu Met Pro 20 25 16 17 PRTArtificial Sequence misc_feature Synthetic peptide 16 Met Gly Leu GlyLeu His Leu Leu Val Leu Ala Ala Ala Leu Gln Gly 1 5 10 15 Ala 17 24 PRTArtificial Sequence misc_feature Synthetic peptide 17 Gly Ala Leu PheLeu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Pro LysSer Lys Arg Lys Val 20 18 9 PRT Artificial Sequence misc_featureSynthetic peptide 18 Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 19 24 PRTArtificial Sequence misc_feature Synthetic peptide 19 Gly Leu Phe GluAla Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile AspGly Gly Gly Tyr Cys 20 20 5 PRT Artificial Sequence misc_feature(1)..(1) Xaa stands for any amino acid 20 Xaa Arg Gly Asp Xaa 1 5 21 27PRT Artificial Sequence misc_feature Synthetic peptide 21 Gly Trp ThrLeu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys AlaLeu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 22 9 PRT Artificial Sequencemisc_feature (1)..(1) Ser stands for optional Serine for coupling 22 SerPhe Cys Tyr Trp Lys Thr Cys Thr 1 5 23 9 PRT Artificial Sequencemisc_feature (1)..(1) Ser stands for optional Serine for coupling 23 SerAsp His Gln Leu Asn Pro Ala Phe 1 5 24 34 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 24 gaguugcuga ugaggccgaaaggccgaaag ucug 34

1. A compound having Formula 107:

wherein X comprises a biologically active molecule; each w independentlycomprises a linker molecule or chemical linkage selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage, y comprises a linker molecule that can bepresent or absent; each R1, R2, R3, and R4 independently comprises O,OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,S-alkylcyano, N or substituted N, and Cholesterol comprises cholesterolor an analog, derivative, or metabolite thereof.
 2. The compound ofclaim 1, wherein said W-Cholesterol comprises a compound having Formula109:

wherein n is independently an integer from about 1 to about
 20. 3. Acompound having Formula 111:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and n is aninteger from about 1 to about
 20. 4. A compound having Formula 114:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and n is aninteger from about 1 to about
 20. 5. A compound having Formula 119:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, each R7independently comprises an acyl group that can be present or absent, andeach n is independently an integer from about 1 to about
 20. 6. Acompound having Formula 121:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, each R7independently comprises an acyl group that can be present or absent, andeach n is independently an integer from about 1 to about
 20. 7. Thecompound of claim 1, wherein X comprises a siNA molecule or a portionthereof.
 8. The compound of claim 3, wherein X comprises a siNA moleculeor a portion thereof.
 9. The compound of claim 4, wherein X comprises asiNA molecule or a portion thereof.
 10. The compound of claim 5, whereinX comprises a siNA molecule or a portion thereof.
 11. The compound ofclaim 6, wherein X comprises a siNA molecule or a portion thereof. 12.The compound of claim 1, wherein each W independently comprises a linkermolecule or chemical linkage selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.
 13. The compound of claim 3, wherein W comprises a linkermolecule or chemical linkage selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.
 14. The compound of claim 4, wherein W comprises a linkermolecule or chemical linkage selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.
 15. The compound of claim 5, wherein W comprises a linkermolecule or chemical linkage selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.
 16. The compound of claim 6, wherein W comprises a linkermolecule or chemical linkage selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.
 17. The compound of claim 7, wherein said siNA moleculecomprises and sense strand and an antisense strand, and wherein saidsense strand is conjugated with a compound comprising Formula
 107. 18.The compound of claim 8, wherein said siNA molecule comprises and sensestrand and an antisense strand, and wherein said sense strand isconjugated with a compound comprising Formula III.
 19. The compound ofclaim 9, wherein said siNA molecule comprises and sense strand and anantisense strand, and wherein said sense strand is conjugated with acompound comprising Formula
 114. 20. The compound of claim 10, whereinsaid siNA molecule comprises and sense strand and an antisense strand,and wherein said sense strand is conjugated with a compound comprisingFormula
 119. 21. The compound of claim 11, wherein said siNA moleculecomprises and sense strand and an antisense strand, and wherein saidsense strand is conjugated with a compound comprising Formula 121.